Here, we report the successful design, construction, and characterization of a 770-kilobase synthetic yeast chromosome II (synII). Our study incorporates characterization at multiple levels-including phenomics, transcriptomics, proteomics, chromosome segregation, and replication analysis-to provide a thorough and comprehensive analysis of a synthetic chromosome. Our Trans-Omics analyses reveal a modest but potentially relevant pervasive up-regulation of translational machinery observed in synII, mainly caused by the deletion of 13 transfer RNAs. By both complementation assays and SCRaMbLE (synthetic chromosome rearrangement and modification by -mediated evolution), we targeted and debugged the origin of a growth defect at 37°C in glycerol medium, which is related to misregulation of the high-osmolarity glycerol response. Despite the subtle differences, the synII strain shows highly consistent biological processes comparable to the native strain.

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BackgroundLignin-derived phenolic compounds are universal in the hydrolysate of pretreated lignocellulosic biomass. The phenolics reduce the efficiency of enzymatic hydrolysis and increase the cost of ethanol production. We investigated inhibition of phenolics on cellulase during enzymatic hydrolysis using vanillin as one of the typical lignin-derived phenolics and Avicel as cellulose substrate.ResultsAs vanillin concentration increased from 0 to 10 mg/mL, cellulose conversion after 72-h enzymatic hydrolysis decreased from 53 to 26 %. Enzyme deactivation and precipitation were detected with the vanillin addition. The enzyme concentration and activity consecutively decreased during hydrolysis, but the inhibition degree, expressed as the ratio of the cellulose conversion without vanillin to the conversion with vanillin (A0/A), was almost independent on hydrolysis time. Inhibition can be mitigated by increasing cellulose loading or cellulase concentration. The inhibition degree showed linear relationship with the vanillin concentration and exponential relationship with the cellulose loading and the cellulase concentration. The addition of calcium chloride, BSA, and Tween 80 did not release the inhibition of vanillin significantly. pH and temperature for hydrolysis also showed no significant impact on inhibition degree. The presence of hydroxyl group, carbonyl group, and methoxy group in phenolics affected the inhibition degree.ConclusionBesides phenolics concentration, other factors such as cellulose loading, enzyme concentration, and phenolic structure also affect the inhibition of cellulose conversion. Lignin-blocking agents have little effect on the inhibition effect of soluble phenolics, indicating that the inhibition mechanism of phenolics to enzyme is likely different from insoluble lignin. The inhibition of soluble phenolics can hardly be entirely removed by increasing enzyme concentration or adding blocking proteins due to the dispersity and multiple binding sites of phenolics than insoluble lignin.Electronic supplementary materialThe online version of this article (doi:10.1186/s13068-016-0485-2) contains supplementary material, which is available to authorized users.

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Furfural and acetic acid are two prevalent inhibitors to microorganisms during cellulosic ethanol production, but molecular mechanisms of tolerance to these inhibitors are still unclear. In this study, genome-wide transcriptional responses to furfural and acetic acid were investigated in Saccharomyces cerevisiae using microarray analysis. We found that 103 and 227 genes were differentially expressed in the response to furfural and acetic acid, respectively. Furfural downregulated genes related to transcriptional control and translational control, while it upregulated stress-responsive genes. Furthermore, furfural also interrupted the transcription of genes involved in metabolism of essential chemicals, such as etrahydrofolate, spermidine, spermine, and riboflavin monophosphate. Acetic acid downregulated genes encoding mitochondrial ribosomal proteins and genes involved in carbohydrate metabolism and regulation and upregulated genes related to amino acid metabolism. The results revealed that furfural and acetic acid had effects on multiple aspects of cellular metabolism on the transcriptional level and that mitochondria might play important roles in response to both furfural and acetic acid. This research has provided insights into molecular response to furfural and acetic acid in S. cerevisiae, and it will be helpful to construct more resistant strains for cellulosic ethanol production.

BackgroundThe oleaginous yeast Yarrowia lipolytica is a promising microbial cell factory due to their biochemical characteristics and native capacity to accumulate lipid-based chemicals. To create heterogenous biosynthesis pathway and manipulate metabolic flux in Y. lipolytica, numerous studies have been done for developing synthetic biology tools for gene regulation. CRISPR interference (CRISPRi), as an emerging technology, has been applied for specifically repressing genes of interest.ResultsIn this study, we established CRISPRi systems in Y. lipolytica based on four different repressors, that was DNase-deactivated Cpf1 (dCpf1) from Francisella novicida, deactivated Cas9 (dCas9) from Streptococcus pyogenes, and two fusion proteins (dCpf1-KRAB and dCas9-KRAB). Ten gRNAs that bound to different regions of gfp gene were designed and the results indicated that there was no clear correlation between the repression efficiency and targeting sites no matter which repressor protein was used. In order to rapidly yield strong gene repression, a multiplex gRNAs strategy based on one-step Golden-brick assembly technology was developed. High repression efficiency 85% (dCpf1) and 92% (dCas9) were achieved in a short time by making three different gRNAs towards gfp gene simultaneously, which avoided the need of screening effective gRNA loci in advance. Moreover, two genes interference including gfp and vioE and three genes repression including vioA, vioB and vioE in protodeoxy-violaceinic acid pathway were also realized.ConclusionTaken together, successful CRISPRi-mediated regulation of gene expression via four different repressors dCpf1, dCas9, dCpf1-KRAB and dCas9-KRAB in Y. lipolytica is achieved. And we demonstrate a multiplexed gRNA targeting strategy can efficiently achieve transcriptional simultaneous repression of several targeted genes and different sites of one gene using the one-step Golden-brick assembly. This timesaving method promised to be a potent transformative tool valuable for metabolic engineering, synthetic biology, and functional genomic studies of Y. lipolytica.Electronic supplementary materialThe online version of this article (10.1186/s12934-018-0909-8) contains supplementary material, which is available to authorized users.

1,1,1-Trichloroethane (TCA), labeled as a priority pollutant by the Environmental Protection Agency (EPA) of China, can be removed from groundwater by sonochemical oxidation. The sonochemical oxidation of TCA in the presence of persulfate (PS) showed a significant synergistic effect. The operational parameters, ultrasonic frequency, PS/TCA molar ratio, radical scavenger, inorganic anions (Cl(-), CO(3)(2-), HCO(3)(-) and NO(3)(-)) and humic acid (HA), were evaluated during the investigation of the sonochemical reaction. The results showed that the degradation of TCA followed pseudo-first-order kinetics, and the rate constant was found to increase with increasing ultrasonic frequency but to decrease with both an increasing PS/TCA molar ratio and an increasing concentration of inorganic anions. With a concentration of 4.46mg/L of HA in solution, an enhanced effect was observed. Further addition of HA retarded the degradation rate of TCA. TCA could be eliminated almost completely by sono-activated persulfate oxidation, with sulfate and hydroxyl radicals serving as the principal oxidants as confirmed by the addition of radical scavengers. Eleven chlorinated degradation intermediates were detected and quantified by purge and trap gas chromatography coupled with mass spectrometry (P&T-GC-MS) in the absence of pH buffer. Three TCA degradation pathways were therefore proposed. In conclusion, the sono-activated persulfate oxidation process appears to be a highly promising technique for the remediation of TCA-contaminated groundwater.

BackgroundThe induction of cellulase production by insoluble carbon source cellulose was a common and efficient strategy, but has some drawbacks, such as difficult fermentation operation, substantial cellulase loss, long fermentation time, and high energy-consumption, resulting in high cost of cellulase production in industry. These drawbacks can be overcome if soluble carbon sources are utilized as the inducers for cellulase production. However, until now the induction efficiency of most soluble carbon sources, especially lactose and glucose, is still inferior to cellulose despite extensive efforts have been made by either optimizing the fermentation process or constructing the recombinant strains. Therefore, strain improvement by metabolic engineering for high induction efficiency of soluble carbon sources is of great interest.Results Trichoderma reesei mutant SEU-7 was constructed from T. reesei RUT-C30 with the overexpression of endogenous gene β-glucosidase (BGL1) by insertional mutagenesis via Agrobacterium tumefaciens-mediated transformation (AMT). Compared to RUT-C30, SEU-7 displays substantially enhanced activities of both cellulase and hemicellulase when grown on either lactose or cellulose. The induction efficiency with lactose was found to be higher than cellulose in strain SEU-7. To the best of our knowledge, we achieved the highest FPase activity in SEU-7 in both batch culture (13.0 IU/mL) and fed-batch culture (47.0 IU/mL) on lactose. Moreover, SEU-7 displayed unrivaled pNPGase activity on lactose in both batch culture (81.0 IU/mL) and fed-batch culture (144.0 IU/mL) as compared to the other reported T. reesei strains in the literature grown in batch or fed-batch experiments on cellulose or lactose. This superiority of SEU-7 over RUT-C30 improves markedly the saccharification ability of SEU-7 on pretreated corn stover. The overexpression of gene BGL1 was found either at the mRNA or at the protein level in the mutant strains with increased cellulase production in comparison with RUT-C30, but only SEU-7 displayed much higher expression of gene BGL1 on lactose than on cellulose. Two copies of gene BGL1 were inserted into the chromosome of T. reesei SEU-7 between KI911141.1:347357 and KI911141.1:347979, replacing the original 623-bp fragment that is not within any genes’ coding region. The qRT-PCR analysis revealed that the mRNA levels of both cellulase and hemicellulase were upregulated significantly in SEU-7, together with the MFS transporter CRT1 and the XYR1 nuclear importer KAP8.ConclusionsRecombinant T. reesei SEU-7 displays hyper-production of both cellulase and hemicellulase on lactose with the highest FPase activity and pNPGase activity for T. reesei, enabling highly efficient saccharification of pretreated biomass. For the first time, the induction efficiency for cellulase production by lactose in T. reesei was reported to be higher than that by cellulose. This outperformance of T. reesei SEU-7, which is strain-specific, is attributed to both the overexpression of gene BGL and the collateral ...