Background: Furfural and acetic acid are the two major inhibitors generated during lignocellulose pretreatment and hydrolysis, would severely inhibit the cell growth, metabolism, and ethanol fermentation efficiency of Zymomonas mobilis. Effective genome shuffling mediated by protoplast electrofusion was developed and then applied to Z. mobilis. Results: After two rounds of genome shuffling, 10 different mutants with improved cell growth and ethanol yield in the presence of 5.0 g/L acetic acid and 3.0 g/L furfural were obtained. The two most prominent genome-shuffled strains, 532 and 533, were further investigated along with parental strains in the presence of 7.0 g/L acetic acid and 3.0 g/L furfural. The results showed that mutants 532 and 533 were superior to the parental strain AQ8-1 in the presence of 7.0 g/L acetic acid, with a shorter fermentation time (30 h) and higher productivity than AQ8-1. Mutant 533 exhibited subtle differences from parental strain F34 in the presence of 3.0 g/L furfural. Mutations present in 10 genome-shuffled strains were identified via whole-genome resequencing, and the source of each mutation was identified as either de novo mutation or recombination of the parent genes. Conclusions: These results indicate that genome shuffling is an efficient method for enhancing stress tolerance in Z. mobilis. The engineered strains generated in this study could be potential cellulosic ethanol producers in the future.

This study aimed to suggest an attention assessment tool using a Digital Pen for measuring the temporal-spatial parameters during the Number Cancelation Test (NCT), and then to establish the normative data for the NCT among children in kindergartens and primary schools in China by recruiting a total of 989 children (496 males). Four measures, i.e., selective attention (SA), speed of cognitive processing (SpC), averaged time of circlings (ATC), and averaged circumference of circled curves (ACCC), were proposed to evaluate the NCT performance. They basically have a development trend with fast speed in the beginning before Grade 1 or 2 of primary schools, and then enter an extremely slow development period (with ceiling or floor effect). SA and SpC have gender and grade main effects, while ATC and ACCC have the grade main effect, only. In particular, females have higher SA scores than males in middle class of kindergarten, and Grade 2–Grade 5 of primary school, but no gender differences in other grades; females have higher SpC scores than males in middle class of kindergarten, and Grade 3–4 of primary school, but no gender differences in other grades. More importantly, in clinical practice, if SA or SpC measure of a child is below than the 5th centile (i.e., p5 level) of his/her grade-specific normative data, then this child may be predicted to have a high-risk of learning disabilities. Findings suggest that the proposed method can be used for early screening of learning disabilities by setting appropriate cut-off values.

Background As one of the clean and sustainable energies, lignocellulosic ethanol has achieved much attention around the world. The production of lignocellulosic ethanol does not compete with people for food, while the consumption of ethanol could contribute to the carbon dioxide emission reduction. However, the simultaneous transformation of glucose and xylose to ethanol is one of the key technologies for attaining cost-efficient lignocellulosic ethanol production at an industrial scale. Genetic modification of strains and constructing consortia were two approaches to resolve this issue. Compared with strain improvement, the synergistic interaction of consortia in metabolic pathways should be more useful than using each one separately. Results In this study, the consortia consisting of suspended Scheffersomyces stipitis CICC1960 and Zymomonas mobilis 8b were cultivated to successfully depress carbon catabolite repression (CCR) in artificially simulated 80G40XRM. With this strategy, a 5.52% more xylose consumption and a 6.52% higher ethanol titer were achieved by the consortium, in which the inoculation ratio between S. stipitis and Z. mobilis was 1:3, compared with the Z. mobilis 8b mono-fermentation. Subsequently, one copy of the xylose metabolic genes was inserted into the Z. mobilis 8b genome to construct Z. mobilis FR2, leading to the xylose final-consumption amount and ethanol titer improvement by 15.36% and 6.81%, respectively. Finally, various corn stover hydrolysates with different sugar concentrations (glucose and xylose 60, 90, 120 g/L), were used to evaluate the fermentation performance of the consortium consisting of S. stipitis CICC1960 and Z. mobilis FR2. Fermentation results showed that a 1.56–4.59% higher ethanol titer was achieved by the consortium compared with the Z. mobilis FR2 mono-fermentation, and a 46.12–102.14% higher ethanol titer was observed in the consortium fermentation when compared with the S. stipitis CICC1960 mono-fermentation. Furthermore, qRT-PCR analysis of xylose/glucose transporter and other genes responsible for CCR explained the reason why the initial ratio inoculation of 1:3 in artificially simulated 80G40XRM had the best fermentation performance in the consortium. Conclusions The fermentation strategy used in this study, i.e., using a genetically modified consortium, had a superior performance in ethanol production, as compared with the S. stipitis CICC1960 mono-fermentation and the Z. mobilis FR2 mono-fermentation alone. This result showed that this strategy has potential for future lignocellulosic ethanol production.

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Objective: This research aimed to provide evidence for the early identification and intervention of children at risk for auditory processing disorder (APD). Electrophysiological studies on children with suspected APDs were systematically reviewed to understand the different electrophysiological characteristics of children with suspected APDs.Methods: Computerized databases such as PubMed, Cochrane, MEDLINE, Web of Science, and EMBASE were searched for retrieval of articles since the establishment of the database through May 18, 2020. Cohort, case-control, and cross-sectional studies that evaluated the literature for the electrophysiological assessment of children with suspected APD were independently reviewed by two researchers for literature screening, literature quality assessment, and data extraction. The Newcastle–Ottawa Scale and 11 entries recommended by the Agency for Healthcare Research and Quality were used to evaluate the quality of the literature.Results: In accordance with the inclusion criteria, 14 articles were included. These articles involved 7 electrophysiological testing techniques: click-evoked auditory brainstem responses, frequency-following responses, the binaural interaction component of the auditory brainstem responses, the middle-latency response, cortical auditory evoked potential, mismatch negativity, and P300. The literature quality was considered moderate.Conclusions: Auditory electrophysiological testing can be used for the characteristic identification of children with suspected APD; however, the value of various electrophysiological testing methods for screening children with suspected APD requires further study.

Panax notoginseng is one of the most famous valuable medical plants in China, and its broad application in clinical treatment has an inseparable relationship with the active molecules, ginsenosides. Ginsenosides are glycoside compounds that have varied structures for the diverse sugar chain. Although extensive work has been done, there are still unknown steps in the biosynthetic pathway of ginsenosides. Here, we screened candidate glycosyltransferase genes based on the previous genome and transcriptome data of P. notoginseng and cloned the full length of 27 UGT genes successfully. Among them, we found that PnUGT33 could catalyze different ginsenoside substrates to produce higher polarity rare ginsenosides by extending the sugar chain. We further analyzed the enzymatic kinetics and predicted the catalytic mechanism of PnUGT33 by simulating molecular docking. After that, we reconstructed the biosynthetic pathway of rare ginsenoside Rg3 and gypenoside LXXV in yeast. By combining the Golden Gate method and overexpressing the UDPG biosynthetic genes, we further improved the yield of engineering yeast strain. Finally, the shake-flask culture yield of Rg3 reached 51 mg/L and the fed-batch fermentation yield of gypenoside LXXV reached 94.5 mg/L, which was the first and highest record.

Acetic acid and furfural (AF) are two major inhibitors of microorganisms during lignocellulosic ethanol production. In our previous study, we successfully engineered Zymomonas mobilis 532 (ZM532) strain by genome shuffling, but the molecular mechanisms of tolerance to inhibitors were still unknown. Therefore, this study investigated the responses of ZM532 and its wild-type Z. mobilis (ZM4) to AF using multi-omics approaches (transcriptomics, genomics, and label free quantitative proteomics). Based on RNA-Seq data, two differentially expressed genes, ZMO_RS02740 (up-regulated) and ZMO_RS06525 (down-regulated) were knocked out and over-expressed through CRISPR-Cas technology to investigate their roles in AF tolerance. Overall, we identified 1865 and 14 novel DEGs in ZM532 and wild-type ZM4. In contrast, 1532 proteins were identified in ZM532 and wild-type ZM4. Among these, we found 96 important genes in ZM532 involving acid resistance mechanisms and survival rates against stressors. Furthermore, our knockout results demonstrated that growth activity and glucose consumption of mutant strains ZM532∆ZMO_RS02740 and ZM4∆ZMO_RS02740 decreased with increased fermentation time from 42 to 55 h and ethanol production up to 58% in ZM532 than that in ZM532∆ZMO_RS02740. Hence, these findings suggest ZMO_RS02740 as a protective strategy for ZM ethanol production under stressful conditions.

Zymomonas mobilis ( Z. mobilis ) is a potential candidate for consolidated bioprocessing (CBP) strain in lignocellulosic biorefinery. However, the low-level secretion of cellulases limits this CBP process, and the mechanism of protein secretion affected by cell wall peptidoglycan is also not well understood. Here we constructed several Penicillin Binding Proteins (PBPs)-deficient strains derivated from Z. mobilis S192 to perturb the cell wall peptidoglycan network and investigated the effects of peptidoglycan on the endoglucanase secretion. Results showed that extracellular recombinant endoglucanase production was significantly enhanced in PBPs mutant strains, notably, △1089/0959 (4.09-fold) and △0959 (5.76-fold) in comparison to parent strains. Besides, for PBPs-deficient strains, the growth performance was not significantly inhibited but with enhanced antibiotic sensitivity and reduced inhibitor tolerance, otherwise, cell morphology was altered obviously. The concentration of intracellular soluble peptidoglycan was increased, especially for single gene deletion. Outer membrane permeability of PBPs-deficient strains was also improved, notably, △1089/0959 (1.14-fold) and △0959 (1.07-fold), which might explain the increased endoglucanase extracellular secretion. Our finding indicated that PBPs-deficient Z. mobilis is capable of increasing endoglucanase extracellular secretion via cell wall peptidoglycan disturbance and it will provide a foundation for the development of CBP technology in Z. mobilis in the future. IMPORTANCE Cell wall peptidoglycan has the function to maintain cell robustness, and also acts as the barrier to secret recombinant proteins from the cytoplasm to extracellular space in Z. mobilis and other bacterias. Herein, we perturb the peptidoglycan synthesis network via knocking out PBPs ( ZMO0197 , ZMO0959 , ZMO1089 ) in order to enhance recombinant endoglycanase extracellular secretion in Z. mobilis S912. This study can not only lay the foundation for understanding the regulatory network of cell wall synthesis but also provide guidance for the construction of CBP strains in Z. mobilis .