Establishment and application of a CRISPR-Cas12a assisted genome-editing system in Zymomonas mobilis.

BACKGROUND: Efficient and convenient genome-editing toolkits can expedite genomic research and strain improvement for desirable phenotypes. Zymomonas mobilis is a highly efficient ethanol-producing bacterium with a small genome size and desirable industrial characteristics, which makes it a promising chassis for biorefinery and synthetic biology studies. While classical techniques for genetic manipulation are available for Z. mobilis, efficient genetic engineering toolkits enabling rapidly systematic and high-throughput genome editing in Z. mobilis are still lacking. RESULTS: Using Cas12a (Cpf1) from Francisella novicida, a recombinant strain with inducible cas12a expression for genome editing was constructed in Z. mobilis ZM4, which can be used to mediate RNA-guided DNA cleavage at targeted genomic loci. gRNAs were then designed targeting the replicons of native plasmids of ZM4 with about 100% curing efficiency for three native plasmids. In addition, CRISPR-Cas12a recombineering was used to promote gene deletion and insertion in one step efficiently and precisely with efficiency up to 90%. Combined with single-stranded DNA (ssDNA), CRISPR-Cas12a system was also applied to introduce minor nucleotide modification precisely into the genome with high fidelity. Furthermore, the CRISPR-Cas12a system was employed to introduce a heterologous lactate dehydrogenase into Z. mobilis with a recombinant lactate-producing strain constructed. CONCLUSIONS: This study applied CRISPR-Cas12a in Z. mobilis and established a genome editing tool for efficient and convenient genome engineering in Z. mobilis including plasmid curing, gene deletion and insertion, as well as nucleotide substitution, which can also be employed for metabolic engineering to help divert the carbon flux from ethanol production to other products such as lactate demonstrated in this work. The CRISPR-Cas12a system established in this study thus provides a versatile and powerful genome-editing tool in Z. mobilis for functional genomic research, strain improvement, as well as synthetic microbial chassis development for economic biochemical production.

Systematic metabolic engineering of Zymomonas mobilis for ß-farnesene production.

Zymomonas mobilis is an ethanologenic bacterium that can produce hopanoids using farnesyl pyrophosphate (FPP), which can be used as the precursor by ß-farnesene synthase for ß-farnesene production. To explore the possibility and bottlenecks of developing Z. mobilis for ß-farnesene production, five heterologous ß-farnesene synthases were selected and screened, and AaBFS from Artemisia annua had the highest ß-farnesene titer. Recombinant strains with AaBFS driven by the strong constitutive promoter Pgap (Pgap-AaBFS) doubled its ß-farnesene production to 25.73 ± 0.31 mg/L compared to the recombinant strain with AaBFS driven by Ptet (Ptet-AaBFS), which can be further improved by overexpressing the Pgap-AaBFS construct using the strategies of multiple plasmids (41.00 ± 0.40 mg/L) or genomic multi-locus integration (48.33 ± 3.40 mg/L). The effect of cofactor NADPH balancing on ß-farnesene production was also investigated, which can be improved only in zwf-overexpressing strains but not in ppnK-overexpressing strains, indicating that cofactor balancing is important and sophisticated. Furthermore, the ß-farnesene titer was improved to 73.30 ± 0.71 mg/L by overexpressing dxs, ispG, and ispH. Finally, the ß-farnesene production was increased to 159.70 ± 7.21 mg/L by fermentation optimization, including the C/N ratio, flask working volume, and medium/dodecane ratio, which was nearly 13-fold improved from the parental strain. This work thus not only generated a recombinant ß-farnesene production Z. mobilis strain but also unraveled the bottlenecks to engineer Z. mobilis for farnesene production, which will help guide the future rational design and construction of cell factories for terpenoid production in non-model industrial microorganisms.

Systematic investigation of TetR-family transcriptional regulators and their roles on lignocellulosic inhibitor acetate tolerance in Zymomonas mobilis.

TetR-family transcriptional regulators are widely distributed among bacteria and involved in various cellular processes such as multidrug and inhibitor resistance. Zymomonas mobilis is a industrial bacterium for lignocellulosic ethanol production. Although TetR-family regulators and their associated RND-family efflux pumps in Z. mobilis have been identified to be differentially expressed under various inhibitors and stressful conditions, there are no systematic investigation yet. In this study, bioinformatic analyses indicated that there are three TetR-family transcriptional regulators (ZMO0281, ZMO0963, ZMO1547) and two RND-family efflux pumps (ZMO0282-0285, ZMO0964-0966) adjacent to corresponding TetR-family regulators of ZMO0281 and ZMO0963 in Z. mobilis. Genetics studies were then carried out with various mutants of TetR-family regulators constructed, and ZMO0281 was characterized to be related to acetate tolerance. Combining transcriptomics and dual-reporter gene system, this study demonstrated that three TetR-family regulators repressed their adjacent genes specifically. Moreover, TetR-family regulator ZMO0281 might also be involved in other cellular processes in the presence of acetate. In addition, the upregulation of RND-family efflux pumps due to ZMO0281 deletion might lead to an energy imbalance and decreased cell growth in Z. mobilis under acetate stress. The systematic investigation of all three TetR-family regulators and their roles on a major lignocellulosic inhibitor acetate tolerance in Z. mobilis thus not only unravels the molecular mechanisms of TetR-family regulators and their potential cross-talks on regulating RND-family efflux pumps and other genes in Z. mobilis, but also provides guidance on understanding the roles of multiple regulators of same family in Z. mobilis and other microorganisms for efficient lignocellulosic biochemical production.

The Effect of Social Appearance Anxiety on the Online Impulse Purchases of Fashionable Outfits Among Female College Students During Pandemic Periods: The Mediating Role of Self-Control and the Moderating Role of Subjective Socioeconomic Status.

Purpose: With the rise of social media, an increasing number of young females have focused more attention on their body image, leading to social appearance anxiety. Thus, the current study aimed to investigate a moderated mediation model of social appearance anxiety and online impulse purchases of fashionable outfits through self-control moderated by subjective socioeconomic status during the pandemic. Methods: A total of 1651 female college students (Age = 17-24, Mage = 19.30, SD = 1.14) with more than one month of closed-off management experience completed self-report questionnaires concerning social appearance anxiety, self-control, online impulse purchases of fashionable outfits, and subjective socioeconomic status. Results: After controlling for the potential influence of coronavirus stress, the results indicated that social appearance anxiety was positively related to online impulse purchases of fashionable outfits among female college students during the pandemic, mediated by self-control. Furthermore, subjective socioeconomic status moderated the indirect link between social appearance anxiety and the online impulse purchase of fashionable outfits. Specifically, subjective socioeconomic status buffers the negative effect of social appearance anxiety on self-control and the risk effect of low self-control on the online impulse purchases of fashionable outfits. Implications: The current study deepens the research on the relationship between social appearance anxiety and online impulse purchases during pandemic periods; meanwhile, it provides evidence for preventing excessive online impulse purchases of fashionable outfits among young females.

The psychological mechanism underlying neuroticism on divorce proneness: The chain mediating roles of negative partner support and couple burnout and the moderating role of economic level.

BACKGROUND: Previous studies have found that neuroticism is a significant predictor of divorce proneness. However, how neuroticism affects divorce proneness remains unclear. Based on the vulnerability-stress-adaptation (VSA) model of marriage, this study aimed to explore the psychological mechanism by which neuroticism affects divorce proneness. METHODS: A total of 752 Chinese heterosexual married individuals were surveyed by the Neurotic Subscale of the NEO Five-Factor Inventory, the Negative Subscale of the Dyadic Coping Inventory, the Couple Burnout Measure, the Divorce Proneness Scale, and the Personal Monthly Income Survey. RESULTS: (1) Negative partner support and couple burnout played chain mediating roles in the relationship between neuroticism and divorce proneness, which constructed a chain mediating model. (2) Economic level played a moderating role in the relationship between couple burnout and divorce proneness, which was the latter part of the chain mediating model. CONCLUSIONS: Divorce proneness can be decreased by perfecting personality traits, reducing negative partner support and couple burnout, and improving the economic level.

Image Comes Dancing With Collaborative Parsing-Flow Video Synthesis.

Transferring human motion from a source to a target person poses great potential in computer vision and graphics applications. A crucial step is to manipulate sequential future motion while retaining the appearance characteristic. Previous work has either relied on crafted 3D human models or trained a separate model specifically for each target person, which is not scalable in practice. This work studies a more general setting, in which we aim to learn a single model to parsimoniously transfer motion from a source video to any target person given only one image of the person, named as Collaborative Parsing-Flow Network (CPF-Net). The paucity of information regarding the target person makes the task particularly challenging to faithfully preserve the appearance in varying designated poses. To address this issue, CPF-Net integrates the structured human parsing and appearance flow to guide the realistic foreground synthesis which is merged into the background by a spatio-temporal fusion module. In particular, CPF-Net decouples the problem into stages of human parsing sequence generation, foreground sequence generation and final video generation. The human parsing generation stage captures both the pose and the body structure of the target. The appearance flow is beneficial to keep details in synthesized frames. The integration of human parsing and appearance flow effectively guides the generation of video frames with realistic appearance. Finally, the dedicated designed fusion network ensure the temporal coherence. We further collect a large set of human dancing videos to push forward this research field. Both quantitative and qualitative results show our method substantially improves over previous approaches and is able to generate appealing and photo-realistic target videos given any input person image. All source code and dataset will be released at https://github.com/xiezhy6/CPF-Net.

Prediction and characterization of promoters and ribosomal binding sites of Zymomonas mobilis in system biology era.

BACKGROUND: Zymomonas mobilis is a model bacterial ethanologen with many systems biology studies reported. Besides lignocellulosic ethanol production, Z. mobilis has been developed as a platform for biochemical production through metabolic engineering. However, identification and rigorous understanding of the genetic origins of cellular function, especially those based in non-coding region of DNA, such as promoters and ribosomal binding sites (RBSs), are still in its infancy. This knowledge is crucial for the effective application of Z. mobilis to new industrial applications of biotechnology for fuels and chemicals production. RESULTS: In this study, we explored the possibility to systematically predict the strength of promoters based on systems biology datasets. The promoter strength was clustered based on the expression values of downstream genes (or proteins) from systems biology studies including microarray, RNA-Seq and proteomics. Candidate promoters with different strengths were selected for further characterization, which include 19 strong, nine medium, and ten weak ones. A dual reporter-gene system was developed which included appropriate reporter genes. These are the opmCherry reporter gene driven by the constitutive PlacUV5 promoter for calibration, and EGFP reporter gene driven by candidate promoters for quantification. This dual reporter-gene system was confirmed using the inducible promoter, Ptet, which was used to determine the strength of these predicted promoters with different strengths. In addition, the dual reporter-gene system was applied to determine four synthetic RBSs with different translation initiation rates based on the prediction from bioinformatics server RBS calculator. Our results showed that the correlations between the prediction and experimental results for the promoter and RBS strength are relatively high, with R 2 values more than 0.7 and 0.9, respectively. CONCLUSIONS: This study not only identified and characterized 38 promoters and four RBSs with different strengths for future metabolic engineering in Z. mobilis, but also established a flow cytometry-based dual reporter-gene system to characterize genetic elements including, but not limited to the promoters and RBSs studied in this work. This study also suggested the feasibility of predicting and selecting candidate genetic elements based on omics datasets and bioinformatics tools. Moreover, the dual reporter-gene system developed in this study can be utilized to characterize other genetic elements of Z. mobilis, which can also be applied to other microorganisms.