Artificial Biosynthetic Pathway for Efficient Synthesis of Vanillin, a Feruloyl-CoA-Derived Natural Product from Eugenol.

Eugenol, the main component of essential oil from the Syzygium aromaticum clove tree, has great potential as an alternative bioresource feedstock for biosynthesis purposes. Although eugenol degradation to ferulic acid was investigated, an efficient method for directly converting eugenol to targeted natural products has not been established. Herein we identified the inherent inhibitions by simply combining the previously reported ferulic acid biosynthetic pathway and vanillin biosynthetic pathway. To overcome this, we developed a novel biosynthetic pathway for converting eugenol into vanillin, by introducing cinnamoyl-CoA reductase (CCR), which catalyzes conversion of coniferyl aldehyde to feruloyl-CoA. This approach bypasses the need for two catalysts, namely coniferyl aldehyde dehydrogenase and feruloyl-CoA synthetase, thereby eliminating inhibition while simplifying the pathway. To further improve efficiency, we enhanced CCR catalytic efficiency via directed evolution and leveraged an artificialvanillin biosensor for high-throughput screening. Switching the cofactor preference of CCR from NADP+ to NAD+ significantly improved pathway efficiency. This newly designed pathway provides an alternative strategy for efficiently biosynthesizing feruloyl-CoA-derived natural products using eugenol.

Transcriptomic analysis reveals hub genes and pathways in response to acetic acid stress in Kluyveromyces marxianus during high-temperature ethanol fermentation.

The thermotolerant yeast Kluyveromyces marxianus is known for its potential in high-temperature ethanol fermentation, yet it suffers from excess acetic acid production at elevated temperatures, which hinders ethanol production. To better understand how the yeast responds to acetic acid stress during high-temperature ethanol fermentation, this study investigated its transcriptomic changes under this condition. RNA sequencing (RNA-seq) was used to identify differentially expressed genes (DEGs) and enriched gene ontology (GO) terms and pathways under acetic acid stress. The results showed that 611 genes were differentially expressed, and GO and pathway enrichment analysis revealed that acetic acid stress promoted protein catabolism but repressed protein synthesis during high-temperature fermentation. Protein-protein interaction (PPI) networks were also constructed based on the interactions between proteins coded by the DEGs. Hub genes and key modules in the PPI networks were identified, providing insight into the mechanisms of this yeast's response to acetic acid stress. The findings suggest that the decrease in ethanol production is caused by the imbalance between protein catabolism and protein synthesis. Overall, this study provides valuable insights into the mechanisms of K. marxianus's response to acetic acid stress and highlights the importance of maintaining a proper balance between protein catabolism and protein synthesis for high-temperature ethanol fermentation.

A corazonin G protein-coupled receptor gene in the tick Ixodes scapularis yields two splice variants, each coding for a specific corazonin receptor.

We have identified a corazonin G protein-coupled receptor (GPCR) gene in the tick Ixodes scapularis, which likely plays a central role in the physiology and behavior of this ectoparasite. This receptor gene is unusually large (1.133 Mb) and yields two corazonin (CRZ) receptor splice variants, where nearly half of the coding regions are exchanged: CRZ-Ra (containing exon 2, exon 3, and exon 4 of the gene) and CRZ-Rb (containing exon 1, exon 3, and exon 4 of the gene). CRZ-Ra codes for a GPCR with a canonical DRF sequence at the border of the third transmembrane helix and the second intracellular loop. The positively-charged R residue from the DRF sequence is important for coupling of G proteins after activation of a GPCR. CRZ-Rb, in contrast, codes for a GPCR with an unusual DQL sequence at this position, still retaining a negatively-charged D residue, but lacking a positively-charged R residue, suggesting different G protein coupling. Another difference between the two splice variants is that exon 2 from CRZ-Ra codes for an N-terminal signal sequence. Normally, GPCRs do not have N-terminal signal sequences, although a few mammalian GPCRs have. In the tick CRZ-Ra, the signal sequence probably assists with inserting the receptor correctly into the RER membrane. We stably transfected Chinese Hamster Ovary cells with each of the two splice variants and carried out bioluminescence bioassays that also included the use of the human promiscuous G protein G16. CRZ-Ra turned out to be selective for I. scapularis corazonin (EC50 = 10-8 M) and could not be activated by related neuropeptides like adipokinetic hormone (AKH) and AKH/corazonin-related peptide (ACP). Similarly, also CRZ-Rb could only be activated by corazonin, although about 4-fold higher concentrations were needed to activate it (EC50 = 4 x 10-8 M). The genomic organization of the tick corazonin GPCR gene is similar to that of the insect AKH and ACP receptor genes. This similar genomic organization can also be found in the human gonadotropin-releasing hormone (GnRH) receptor gene, confirming previous conclusions that the corazonin, AKH, and ACP receptor genes are the true arthropod orthologues of the human GnRH receptor gene.

Silage Fermentation on Sweet Sorghum Whole Plant for Fen-Flavor Baijiu.

The technology for producing bioethanol from sweet sorghum stalks by solid-state fermentation has developed rapidly in recent years, and has many similarities with traditional Chinese liquor production. However, the product from sweet sorghum stalks was lacking in volatile flavors, and the level of harmful contents were uncertain, therefore it could not be sold as liquor. In this study, the protein, fat, and tannin in the clusters and leaves of sweet sorghum were utilized to increase the content of flavor compounds in the ethanol product through the anaerobic fermentation of Saccharomyces cerevisiae. Meanwhile, the silage fermentation method was used to extend the preservation time of the raw materials and to further enhance the flavors of Fen-flavor liquor, with ethyl acetate as the characteristic flavor. The effects of different feedstock groups on ethyl acetate, ethyl lactate, methanol, acetaldehyde, acetal, fusel oil, total acid, and total ester were evaluated by analyzing the chemical composition of different parts of sweet sorghum and determined by gas chromatograph. The effect of different fermentation periods on the volatile flavor of sweet sorghum Baijiu was evaluated. The yield of the characteristic volatile flavor was increased by the extension of the fermentation time. Sweet sorghum Baijiu with a high ester content can be used as a flavoring liquor, blended with liquor with a shorter fermentation period to prepare the finished Fen-flavor Baijiu, conforming to the Chinese national standard for sale.

Expression level of miRNA in the peripheral blood of patients with multiple myeloma and its clinical significance.

OBJECTIVE: To investigate the expression level of serum miRNA-192-5p and its clinical value in the diagnosis and care of patients with multiple myeloma (MM). METHODS: Eighty-eight patients with MM admitted to our hospital from June 2017 to April 2020 were selected as the observation group. In addition, 70 patients who received osteoporosis testing in our hospital in the corresponding period but were excluded from having MM and haematological malignancy were selected as the control group. The relative expression level of serum miRNA-192-5p was detected. The expression level of serum miRNA and its correlation with patient-related clinical parameters were compared and analyzed. The ROC curve was used to analyze its diagnostic efficacy for MM. RESULTS: The relative expression level of serum miRNA-192-5p in MM patients was remarkably lower than that in the control group (P < 0.05); the AUC area of serum miRNA-192-5p in patients with a diagnosis of MM was 0.853, with a cutoff value of 0.72, the sensitivity of 86.30%, and the specificity of 81.20%, P = 0.030. The relative expression level of miRNA-192-5p in the serum of patients with high ß2-MG and creatinine levels was markedly reduced compared to that in patients with low ß2-MG levels (P < 0.05); the relative expression level of miRNA-192-5p in the serum of patients with low hemoglobin and albumin levels was markedly reduced compared to that in patients with normal hemoglobin and albumin (P < 0.05); and there was significantly negative correlation between the relative expression level of miRNA-192-5p in the serum of MM patients and IgG and IgA levels, respectively (P < 0.05). CONCLUSION: miRNA-192-5p may serve as an auxiliary diagnostic tool in the diagnosis of MM. Furthermore, because there is certain correlation between serum miRNA-192-5p and MM progression and prognosis, it may be regarded as a novel marker for MM monitoring.

De Novo Biosynthesis of Chlorogenic Acid Using an Artificial Microbial Community.

Engineering an artificial microbial community for natural product production is a promising strategy. As mono- and dual-culture systems only gave non-detectable or minimal chlorogenic acid (CGA) biosynthesis, here, a polyculture of three recombinant Escherichia coli strains, acting as biosynthetic modules of caffeic acid (CA), quinic acid (QA), and CGA, was designed and used for de novo CGA biosynthesis. An influx transporter of 3-dehydroshikimic acid (DHS)/shikimic acid (SA), ShiA, was introduced into the QA module-a DHS auxotroph. The QA module proportion in the polyculture and CGA production were found to be dependent on ShiA expression, providing an alternative approach for controlling microbial community composition. The polyculture strategy avoids metabolic flux competition in the biosynthesis of two CGA precursors, CA and QA, and allows production improvement by balancing module proportions. The performance of this polyculture approach was superior to that of previously reported approaches of de novo CGA production.

Overexpression of SFA1 in engineered Saccharomyces cerevisiae to increase xylose utilization and ethanol production from different lignocellulose hydrolysates.

Here, an engineered Saccharomyces cerevisiae strain SFA1OE was constructed by overexpressing SFA1 in a reported WXY70 with effective six-gene clusters. Under simulated maize hydrolysate, SFA1OE produced an ethanol yield of 0.492 g/g totalsugars within 48 h. The productivity of SFA1OE was comprehensively evaluated in typical hydrolysates from stalks of maize, sweet sorghum, wheat and Miscanthus. Within 48 h, SFA1OE achieved an ethanol yield of 0.489 g/g totalsugars in the optimized hydrolysate of alkaline-distilled sweet sorghum bagasse derived from Advanced Solid-State Fermentation process. By crossing SFA1OE with a DQ1-derived haploid strain, we obtained an evolved diploid strain SQ-2, exhibiting improved ethanol production and thermotolerance. This study demonstrates that overexpressing SFA1 enables efficient fermentation performance in different lignocellulosic hydrolysates, especially in the hydrolysate of alkaline-distilled sweet sorghum bagasse. The increased cellulosic bioethanol production of SFA1OE provides a promising platform for efficient biorefineries.

Distinct structural modulation of photosystem I and lipid environment stabilizes its tetrameric assembly.

Photosystem I (PSI) is able to form different oligomeric states across various species. To reveal the structural basis for PSI dimerization and tetramerization, we structurally investigated PSI from the cyanobacterium Anabaena. This revealed a disrupted trimerization domain due to lack of the terminal residues of PsaL in the lumen, which resulted in PSI dimers with loose connections between monomers and weaker energy-coupled chlorophylls than in the trimer. At the dimer surface, specific phospholipids, cofactors and interactions in combination facilitated recruitment of another dimer to form a tetramer. Taken together, the relaxed luminal connections and lipid specificity at the dimer interface account for membrane curvature. PSI tetramer assembly appears to increase the surface area of the thylakoid membrane, which would contribute to PSI crowding.

Proteomic profiling and integrated analysis with transcriptomic data bring new insights in the stress responses of Kluyveromyces marxianus after an arrest during high-temperature ethanol fermentation.

BACKGROUND: The thermotolerant yeast Kluyveromyces marxianus is a potential candidate for high-temperature fermentation. When K. marxianus was used for high-temperature ethanol fermentation, a fermentation arrest was observed during the late fermentation stage and the stress responses have been investigated based on the integration of RNA-Seq and metabolite data. In order to bring new insights into the cellular responses of K. marxianus after the fermentation arrest during high-temperature ethanol fermentation, quantitative proteomic profiling and integrated analysis with transcriptomic data were performed in this study. RESULTS: Samples collected at 14, 16, 18, 20 and 22 h during high-temperature fermentation were subjected to isobaric tags for relative and absolute quantitation (iTRAQ)-based proteomic profiling and integrated analysis with transcriptomic data. The correlations between transcripts and proteins for the comparative group 16 h vs 14 h accounted for only 4.20% quantified proteins and 3.23% differentially expressed proteins (DEPs), respectively, much higher percentages of correlations (30.56%-59.11%) were found for other comparative groups (i.e., 18 h vs 14 h, 20 h vs 14 h, and 22 h vs 14 h). According to Spearman correlation tests between transcriptome and proteome (the absolute value of a correlation coefficient between 0.5 and 1 indicates a strong correlation), poor correlations were found for all quantified proteins (R = - 0.0355 to 0.0138), DEPs (R = - 0.0079 to 0.0233) and the DEPs with opposite expression trends to corresponding differentially expressed genes (DEGs) (R = - 0.0478 to 0.0636), whereas stronger correlations were observed in terms of the DEPs with the same expression trends as the correlated DEGs (R = 0.5593 to 0.7080). The results of multiple reaction monitoring (MRM) verification indicate that the iTRAQ results were reliable. After the fermentation arrest, a number of proteins involved in transcription, translation, oxidative phosphorylation and fatty acid metabolism were down-regulated, some molecular chaperones and proteasome proteins were up-regulated, the ATPase activity significantly decreased, and the total fatty acids gradually accumulated. In addition, the contents of palmitic acid, oleic acid, C16, C18, C22 and C24 fatty acids increased by 16.77%, 28.49%, 14.14%, 26.88%, 628.57% and 125.29%, respectively. CONCLUSIONS: This study confirmed some biochemical and enzymatic alterations provoked by the stress conditions in the specific case of K. marxianus: such as decreases in transcription, translation and oxidative phosphorylation, alterations in cellular fatty acid composition, and increases in the abundance of molecular chaperones and proteasome proteins. These findings provide potential targets for further metabolic engineering towards improvement of the stress tolerance in K. marxianus.

Understanding the stress responses of Kluyveromyces marxianus after an arrest during high-temperature ethanol fermentation based on integration of RNA-Seq and metabolite data.

The thermotolerant Kluyveromyces marxianus is a potential candidate for high-temperature ethanol fermentation. Although K. marxianus exhibited high ethanol productivity at 45 °C during the early fermentation stage, we observed a fermentation arrest due to the accumulated inhibitors. The stress responses of K. marxianus during high-temperature fermentation were revealed based on integration of RNA sequencing (RNA-Seq) and metabolite data. High temperature stimulated mitochondrial respiration but repressed the tricarboxylic acid (TCA) cycle, leading to increased generation of reactive oxygen species (ROS) and a lowered ratio of reduced nicotinamide adenine dinucleotide (NADH)/oxidized nicotinamide adenine dinucleotide (NAD+). Glycerol production was enhanced during the early fermentation stage, which might contribute to NADH reoxidation and ROS generation. Excess ROS could be neutralized by reduced nicotinamide adenine dinucleotide phosphate (NADPH) that might be reserved in the following ways: (1) decreased biosynthesis of branched-chain amino acids (BCAAs) reduced NADPH consumption; (2) enhanced acetic acid production increased NADPH regeneration. The degree of fatty acid unsaturation was also reduced to adapt to high temperature. In addition, stress responses were also observed after the fermentation arrest at 45 °C. Genes related to peroxidase activity, iron-sulfur cluster assembly, and flavin mononucleotide (FMN) binding were downregulated, while genes associated with DNA repair and lipid composition of the plasma were upregulated. The yeast also produced more ergosterol to deal with ethanol stress. This study gains comprehensive insights into the K. marxianus transcriptome under various stresses during high-temperature ethanol fermentation, providing rich information for further metabolic engineering towards improved stress tolerance and ethanol production.

CRISPR/Cas-based screening of a gene activation library in Saccharomyces cerevisiae identifies a crucial role of OLE1 in thermotolerance.

CRISPR/Cas-based (clustered regularly interspaced short palindromic repeats/CRISPR-associated) screening has been proved to be an efficient method to study functional genomics from yeast to human. In this study, we report the development of a focused CRISPR/Cas-based gene activation library in Saccharomyces cerevisiae and its application in gene identification based on functional screening towards improved thermotolerance. The gene activation library was subjected to screening at 42°C, and the same library cultured at 30°C was set as a control group. After five successive subcultures, five clones were randomly picked from the libraries cultured at 30 and 42°C, respectively. The five clones selected at 30°C contain the specificity sequences of five different single guide RNAs, whereas all the five clones selected at 42°C contain the specificity sequence of one sgRNA that targets the promoter region of OLE1. A crucial role of OLE1 in thermotolerance was identified: the overexpression of OLE1 increased fatty acid unsaturation, and thereby helped counter lipid peroxidation caused by heat stress, rendering the yeast thermotolerant. This study described the application of CRISPR/Cas-based gene activation screening with an example of thermotolerant yeast screening, demonstrating that this method can be used to identify functional genes in yeast.

Engineering TATA-binding protein Spt15 to improve ethanol tolerance and production in Kluyveromyces marxianus.

BACKGROUND: Low ethanol tolerance of Kluyveromyces marxianus limits its application in high-temperature ethanol fermentation. As a complex phenotype, ethanol tolerance involves synergistic actions of many genes that are widely distributed throughout the genome, thereby being difficult to engineer. TATA-binding protein is the most common target of global transcription machinery engineering for improvement of complex phenotypes. RESULTS: A random mutagenesis library of K. marxianus TATA-binding protein Spt15 was constructed and subjected to screening under ethanol stress. Two mutant strains with improved ethanol tolerance were identified, one of which (denoted as M2) exhibited increased ethanol productivity. The mutant of Spt15 in strain M2 (denoted as Spt15-M2) has a single amino acid substitution at position 31 (Lys → Glu). RNA-Seq-based transcriptomic analysis revealed cellular transcription profile changes resulting from Spt15-M2. Spt15-M2 caused changes in transcriptional level of most of the genes in the central carbon metabolism network. Compared with control strain, 444 differentially expressed genes (DEGs) were identified in strain M2 (fold change > 2, Padj < 0.05), including 48 up-regulated and 396 down-regulated. The up-regulated DEGs are involved in amino acid transport, long-chain fatty acid biosynthesis and MAPK signaling pathway, while the down-regulated DEGs are related to ribosome biogenesis, translation and protein synthesis. Five candidate genes (GAP1, GNP1, FAR1, STE2 and TEC1), which were found to be up-regulated in M2 strain, were overexpressed for a gain-of-function assay. However, the overexpression of no single gene helped improve ethanol tolerance as SPT15-M2 did. CONCLUSIONS: This work demonstrates that ethanol tolerance of K. marxianus can be improved by engineering its TATA-binding protein. A single amino acid substitution (K31E) of TATA-binding protein Spt15 is able to bring differential expression of hundreds of genes that acted as an interconnected network for the phenotype of ethanol tolerance. Future perspectives of this technique in K. marxianus were discussed.

Signature pathway expression of xylose utilization in the genetically engineered industrial yeast Saccharomyces cerevisiae.

Haploid laboratory strains of Saccharomyces cerevisiae are commonly used for genetic engineering to enable their xylose utilization but little is known about the industrial yeast which is often recognized as diploid and as well as haploid and tetraploid. Here we report three unique signature pathway expression patterns and gene interactions in the centre metabolic pathways that signify xylose utilization of genetically engineered industrial yeast S. cerevisiae NRRL Y-50463, a diploid yeast. Quantitative expression analysis revealed outstanding high levels of constitutive expression of YXI, a synthesized yeast codon-optimized xylose isomerase gene integrated into chromosome XV of strain Y-50463. Comparative expression analysis indicated that the YXI was necessary to initiate the xylose metabolic pathway along with a set of heterologous xylose transporter and utilization facilitating genes including XUT4, XUT6, XKS1 and XYL2. The highly activated transketolase and transaldolase genes TKL1, TKL2, TAL1 and NQM1 as well as their complex interactions in the non-oxidative pentose phosphate pathway branch were critical for the serial of sugar transformation to drive the metabolic flow into glycolysis for increased ethanol production. The significantly increased expression of the entire PRS gene family facilitates functions of the life cycle and biosynthesis superpathway for the yeast. The outstanding higher levels of constitutive expression of YXI and the first insight into the signature pathway expression and the gene interactions in the closely related centre metabolic pathways from the industrial yeast aid continued efforts for development of the next-generation biocatalyst. Our results further suggest the industrial yeast is a desirable delivery vehicle for new strain development for efficient lignocellulose-to-advanced biofuels production.

Comparative transcriptome combined with morpho-physiological analyses revealed key factors for differential cadmium accumulation in two contrasting sweet sorghum genotypes.

Cadmium (Cd) is a widespread soil contaminant threatening human health. As an ideal energy plant, sweet sorghum (Sorghum bicolor (L.) Moench) has great potential in phytoremediation of Cd-polluted soils, although the molecular mechanisms are largely unknown. In this study, key factors responsible for differential Cd accumulation between two contrasting sweet sorghum genotypes (high-Cd accumulation one H18, and low-Cd accumulation one L69) were investigated. H18 exhibited a much higher ability of Cd uptake and translocation than L69. Furthermore, Cd uptake through symplasmic pathway and Cd concentrations in xylem sap were both higher in H18 than those in L69. Root anatomy observation found the endodermal apoplasmic barriers were much stronger in L69, which may restrict the Cd loading into xylem. The molecular mechanisms underlying these morpho-physiological traits were further dissected by comparative transcriptome analysis. Many genes involved in cell wall modification and heavy metal transport were found to be Cd-responsive DEGs and/or DEGs between these two genotypes. KEGG pathway analysis found phenylpropanoid biosynthesis pathway was over-represented, indicating this pathway may play important roles in differential Cd accumulation between two genotypes. Based on these results, a schematic representation of main processes involved in differential Cd uptake and translocation in H18 and L69 is proposed, which suggests that higher Cd accumulation in H18 depends on a multilevel coordination of efficient Cd uptake and transport, including efficient root uptake and xylem loading, less root cell wall binding, and weaker endodermal apoplasmic barriers.

[Construction of high-yield strain by optimizing lycopene cyclase for ß-carotene production].

To optimize key enzymes, such as to explore the gene resources and to modify the expression level, can maximize metabolic pathways of target products. ß-carotene is a terpenoid compound with important application value. Lycopene cyclase (CrtY) is the key enzyme in ß-carotene biosynthesis pathway, catalyzing flavin adenine dinucleotide (FAD)-dependent cyclization reaction and ß-carotene synthesis from lycopene precursor. We optimized lycopene cyclase (CrtY) to improve the synthesis of ß-carotene and determined the effect of CrtY expression on metabolic pathways. Frist, we developed a ß-carotene synthesis module by coexpressing the lycopene ß-cyclase gene crtY with crtEBI module in Escherichia coli. Then we simultaneously optimized the ribosome-binding site (RBS) intensity and the species of crtY using oligo-linker mediated DNA assembly method (OLMA). Five strains with high ß-carotene production capacity were screened out from the OLMA library. The ß-carotene yields of these strains were up to 15.79-18.90 mg/g DCW (Dry cell weight), 65% higher than that of the original strain at shake flask level. The optimal strain CP12 was further identified and evaluated for ß-carotene production at 5 L fermentation level. After process optimization, the final ß-carotene yield could reach to 1.9 g/L. The results of RBS strength and metabolic intermediate analysis indicated that an appropriate expression level of CrtY could be beneficial for the function of the ß-carotene synthesis module. The results of this study provide important insight into the optimization of ß-carotene synthesis pathway in metabolic engineering.

The transcription factors Hsf1 and Msn2 of thermotolerant Kluyveromyces marxianus promote cell growth and ethanol fermentation of Saccharomyces cerevisiae at high temperatures.

BACKGROUND: High temperature inhibits cell growth and ethanol fermentation of Saccharomyces cerevisiae. As a complex phenotype, thermotolerance usually involves synergistic actions of many genes, thereby being difficult to engineer. The overexpression of either endogenous or exogenous stress-related transcription factor genes in yeasts was found to be able to improve relevant stress tolerance of the hosts. RESULTS: To increase ethanol yield of high-temperature fermentation, we constructed a series of strains of S. cerevisiae by expressing 8 transcription factor genes from S. cerevisiae and 7 transcription factor genes from thermotolerant K. marxianus in S. cerevisiae. The results of growth curve measurements and spotting test show that KmHsf1 and KmMsn2 can enhance cell growth of S. cerevisiae at 40-42 °C. According to the results of batch fermentation at 43 °C with an initial glucose concentration of 104.8 g/l, the fermentation broths of KmHSF1 and KmMSN2-expressing strains could reach final ethanol concentrations of 27.2 ± 1.4 and 27.6 ± 1.2 g/l, respectively, while the control strain just produced 18.9 ± 0.3 g/l ethanol. Transcriptomic analysis found that the expression of KmHSF1 and KmMSN2 resulted in 55 (including 31 up-regulated and 24 down-regulated) and 50 (including 32 up-regulated and 18 down-regulated) genes with different expression levels, respectively (padj < 0.05). The results of transcriptomic analysis also reveal that KmHsf1 might increase ethanol production by regulating genes related to transporter activity to limit excessive ATP consumption and promote the uptake of glucose; while KmMsn2 might promote ethanol fermentation by regulating genes associated with glucose metabolic process and glycolysis/gluconeogenesis. In addition, KmMsn2 might also help to cope with high temperature by regulating genes associated with lipid metabolism to change the membrane fluidity. CONCLUSIONS: The transcription factors KmHsf1 and KmMsn2 of thermotolerant K. marxianus can promote both cell growth and ethanol fermentation of S. cerevisiae at high temperatures. Different mechanisms of KmHsf1 and KmMsn2 in promoting high-temperature ethanol fermentation of S. cerevisiae were revealed by transcriptomic analysis.

Identification for the capability of Cd-tolerance, accumulation and translocation of 96 sorghum genotypes.

Cadmium (Cd) pollution is a worldwide environmental problem which heavily threatens human health and food security. Sorghum, as one of the most promising energy crop, has been considered to be the source of high-quality feedstock for ethanol fuel. Ninety-six sorghum genotypes were investigated under hydroponic conditions to compare their capabilities of Cd-tolerance, accumulation and translocation for their potential in remediation of Cd contamination. Different genotypes varied largely in the tolerance to Cd stress with tolerance indexes ranked from 0.107 to 0.933. Great difference was also found in Cd uptake and accumulation with concentrations ranging from 19.0 to 202.4mg/kg in shoots and 277.0-898.3mg/kg in roots. The total amounts of Cd ranked from 6.1 to 25.8µg per plant and the highest translocation factor was over 4 times higher than the lowest one. The correlation analysis demonstrated that Cd concentration in shoot reflected the ability of Cd translocation and tolerance of sorghum, and the path coefficient analysis indicated that root biomass could be taken as a biomarker to evaluate Cd extraction ability of sorghum. The results in this study can facilitate the restoring of Cd contaminated areas by sorghum.

Genome-Wide Identification of Sorghum bicolor Laccases Reveals Potential Targets for Lignin Modification.

Laccase is a key enzyme in plant lignin biosynthesis as it catalyzes the final step of monolignols polymerization. Sweet sorghum [Sorghum bicolor (L.) Moench] is considered as an ideal feedstock for ethanol production, but lignin greatly limits the production efficiency. No comprehensive analysis on laccase has ever been conducted in S. bicolor, although it appears as the most promising target for engineering lignocellulosic feedstock. The aim of our work is to systematically characterize S. bicolor laccase gene family and to identify the lignin-specific candidates. A total of twenty-seven laccase candidates (SbLAC1-SbLAC27) were identified in S. bicolor. All SbLACs comprised the equivalent L1-L4 signature sequences and three typical Cu-oxidase domains, but exhibited diverse intron-exon patterns and relatively low sequence identity. They were divided into six groups by phylogenetic clustering, revealing potential distinct functions, while SbLAC5 was considered as the closest lignin-specific candidate. qRT-PCR analysis deciphered that SbLAC genes were expressed preferentially in roots and young internodes of sweet sorghum, and SbLAC5 showed high expression, adding the evidence that SbLAC5 was bona fide involved in lignin biosynthesis. Besides, high abundance of SbLAC6 transcripts was detected, correlating it a potential role in lignin biosynthesis. Diverse cis regulatory elements were recognized in SbLACs promoters, indicating putative interaction with transcription factors. Seven SbLACs were found to be potential targets of sbi-miRNAs. Moreover, putative phosphorylation sites in SbLAC sequences were identified. Our research adds to the knowledge for lignin profile modification in sweet sorghum.

Adipokinetic hormones and their G protein-coupled receptors emerged in Lophotrochozoa.

Most multicellular animals belong to two evolutionary lineages, the Proto- and Deuterostomia, which diverged 640-760 million years (MYR) ago. Neuropeptide signaling is abundant in animals belonging to both lineages, but it is often unclear whether there exist evolutionary relationships between the neuropeptide systems used by proto- or deuterostomes. An exception, however, are members of the gonadotropin-releasing hormone (GnRH) receptor superfamily, which occur in both evolutionary lineages, where GnRHs are the ligands in Deuterostomia and GnRH-like peptides, adipokinetic hormone (AKH), corazonin, and AKH/corazonin-related peptide (ACP) are the ligands in Protostomia. AKH is a well-studied insect neuropeptide that mobilizes lipids and carbohydrates from the insect fat body during flight. In our present paper, we show that AKH is not only widespread in insects, but also in other Ecdysozoa and in Lophotrochozoa. Furthermore, we have cloned and deorphanized two G protein-coupled receptors (GPCRs) from the oyster Crassostrea gigas (Mollusca) that are activated by low nanomolar concentrations of oyster AKH (pQVSFSTNWGSamide). Our discovery of functional AKH receptors in molluscs is especially significant, because it traces the emergence of AKH signaling back to about 550 MYR ago and brings us closer to a more complete understanding of the evolutionary origins of the GnRH receptor superfamily.

Morphophysiological characteristic analysis demonstrated the potential of sweet sorghum (Sorghum bicolor (L.) Moench) in the phytoremediation of cadmium-contaminated soils.

Cadmium (Cd) contamination is a worldwide environmental problem, and remediation of Cd pollution is of great significance for food production as well as human health. Here, the responses of sweet sorghum cv. 'M-81E' to cadmium stress were studied for its potential as an energy plant in restoring soils contaminated by cadmium. In hydroponic experiments, the biomass of 'M-81E' showed no obvious change under 10 µM cadmium treatment. Cadmium concentration was the highest in roots of seedlings as well as mature plants, but in agricultural practice, the valuable and harvested parts of sweet sorghum are shoots, so promoting the translocation of cadmium to shoots is of great importance in order to improve its phytoremediation capacity. Further histochemical assays with dithizone staining revealed that cadmium was mainly concentrated in the stele of roots and scattered in intercellular space of caulicles. Moreover, the correlation analysis showed that Cd had a negative relationship with iron (Fe), zinc (Zn), and manganese (Mn) in caulicles and leaves and a positive relationship with Fe in roots. These results implied that cadmium might compete with Fe, Zn, and Mn for the transport binding sites and further prevent their translocation to shoots. In addition, transmission electron microscopic observations showed that under 100 µM cadmium treatment, the structure of chloroplast was impaired and the cell wall of vascular bundle cells in leaves and xylem and phloem cells in roots turned thicker compared to control. In summary, morphophysiological characteristic analysis demonstrated sweet sorghum can absorb cadmium and the growth is not negatively affected by mild level cadmium stress; thus, it is a promising material for the phytoremediation of cadmium-contaminated soils considering its economic benefit. This study also points out potential strategies to improve the phytoremediation capacity of sweet sorghum through genetic modification of transporters and cell wall components.