Weak Promoter Research Articles

Construction and application of a synthetic promoter library for Corynebacterium glutamicum

Promoter is an important genetic tool for fine-tuning of gene expression and has been widely used for metabolic engineering. Corynebacterium glutamicum is an important chassis for industrial biotechnology. However, promoter libraries that are applicable to C. glutamicum have been rarely reported, except for a few developed based on synthetic sequences containing random mutations. In this study, we constructed a promoter library based on the native promoter of odhA gene by mutating the -10 region and the bystanders. Using a red fluorescent protein (RFP) as the reporter, 57 promoter mutants were screened by fluorescence imaging technology in a high-throughput manner. These mutants spanned a strength range between 2.4-fold and 19.6-fold improvements of the wild-type promoter. The strongest mutant exhibited a 2.3-fold higher strength than the widely used strong inducible promoter Ptrc. Sequencing of all 57 mutants revealed that 55 mutants share a 1-4 bases shift (4 bases shift for 68% mutants) of the conserved -10 motif "TANNNT" to the 3' end of the promoter, compared to the wild-type promoter. Conserved T or G bases at different positions were observed for strong, moderate, and weak promoter mutants. Finally, five promoter mutants with different strength were employed to fine-tune the expression of γ-glutamyl kinase (ProB) for L-proline biosynthesis. Increased promoter strength led to enhanced L-proline production and the highest L-proline titer of 6.4 g/L was obtained when a promoter mutant with a 9.8-fold higher strength compared to the wild-type promoter was used for ProB expression. The use of stronger promoter variants did not further improve L-proline production. In conclusion, a promoter library was constructed based on a native C. glutamicum promoter PodhA. The new promoter library should be useful for systems metabolic engineering of C. glutamicum. The strategy of mutating native promoter may also guide the construction of promoter libraries for other microorganisms.

Synthetic Biology Toolkit for Marker-Less Integration of Multigene Pathways into Pichia pastoris via CRISPR/Cas9.

Pichia pastoris, an important methylotrophic yeast, is currently mainly used for the expression of recombinant proteins and has great potential applications in the production of value-added compounds (e.g., chemical and natural products). However, the construction of P. pastoris cell factories is largely hindered by the lack of genetic tools for the manipulation of multigene biosynthetic pathways. Therefore, the present study aimed to establish a CRISPR-based synthetic biology toolkit for the integration and assembly of multigene biosynthetic pathways into the chromosome of P. pastoris. First, 23 intergenic regions were selected and characterized as potential integration sites, with a focus on the integration efficiency and heterologous gene expression levels. In addition, a panel of constitutive and methanol-inducible promoters with different strengths (weak, medium, and strong promoters) were characterized to control the expression of biosynthetic pathway genes to the desirable levels. With a series of gRNA plasmids (for single-locus, two-loci, and three-loci integration) and donor plasmids (containing homology arms for integration and promoters and terminators for driving heterologous gene expression) as major components, a CRISPR-based synthetic biology toolkit was established, which enabled the integration of one locus, two loci, and three loci with efficiencies as high as ∼100, ∼93, and ∼75%, respectively, in P. pastoris GS115 strain. Finally, the application of the toolkit was demonstrated by the construction of a series of P. pastoris cell factories, which could produce 2,3-butanediol, β-carotene, zeaxanthin, and astaxanthin with methanol as the sole carbon and energy source. The P. pastoris synthetic biology toolkit is highly standardized and can be employed to construct P. pastoris cell factories with high efficiency.

Antimicrobial Resistance and Molecular Characterization of Gene Cassettes from Class 1 Integrons in Escherichia coli Strains.

To investigate the antimicrobial resistance and molecular characterization of gene cassettes from class 1 integrons in Escherichia coli strains isolated from hospitalized patients. Bacterial identification was conducted using the Vitek-2 Compact system, and antimicrobial susceptibility analysis was performed using the Kirby-Bauer method. Class 1 integrons, integrase genes, the variable regions of integrons and promoters from the isolated E. coli were screened by polymerase chain reaction, and subjected to DNA sequencing. In total, 138 E. coli strains were collected from the hospitalized patients, most from urine specimens (41.30%, 57/138). Antimicrobial resistance to ampicillin (89.86%) was most prevalent, with 79.99% of strains being multidrug-resistant (MDR). The class 1 integron integrase intI1 gene was detected in 67.39% of the isolates (93/138). Three gene cassette arrays and 5 antimicrobial resistance gene cassettes were detected in 69 of the class 1 integron-positive strains. The most common gene cassette array was dfrA17-aadA5. Of the 93 intI1-positive strains, 5 different common promoters were detected. The most prevalent common promoter was PcH1, and most isolates contained the dfrA17-aadA5 gene cassette array. In summary, antimicrobial resistance and MDR were prevalent among E. coli isolates in our city Weifang in Shandong Provence China. Gene cassettes of the class 1 integron variable region mostly conferred resistance to traditional antimicrobials. Weak promoters in the variable regions were predominant in this study. Integrons pose a great threat to the treatment of MDR bacterial infections and further investigations are needed.

Transcriptional Organization of the Salmonella Typhimurium Phage P22 pid ORFan Locus.

Many phage genes lack sequence similarity to any other open reading frame (ORF) in current databases. These enigmatic ORFan genes can have a tremendous impact on phage propagation and host interactions but often remain experimentally unexplored. We previously revealed a novel interaction between phage P22 and its Salmonella Typhimurium host, instigated by the ORFan gene pid (for phage P22 encoded instigator of dgo expression) and resulting in derepression of the host dgoRKAT operon. The pid gene is highly expressed in phage carrier cells that harbor a polarly located P22 episome that segregates asymmetrically among daughter cells. Here, we discovered that the pid locus is fitted with a weak promoter, has an exceptionally long 5′ untranslated region that is instructive for a secondary pid mRNA species, and has a 3′ Rho-independent termination loop that is responsible for stability of the pid transcript.

Has the Sustainable Development Planning Policy Promoted the Green Transformation in China's Resource-based Cities?

Scientific evaluation of the transformation effect of resource-based cities is an important way to measure the effect of the implementation of national transformation policies, and it is also an important issue in the study of China's urban transformation development under the "30-60" goal of carbon peaking and carbon neutral. Based on the systematic understanding of the connotation of green transformation development in resource-based cities, this study constructed a framework for evaluating the green transformation performance (GTP) of resource-based cities in China, measured the GTP of 115 prefecture-level and above resource-based cities in China using the entropy weight-TOPSIS model, and further quantitatively assessed the policy effects of sustainable development planning (SDP) in China's resource-based cities from both the overall and local levels using synthetic control method. The results showed the following. (1) Time-series evolution characteristics analysis results showed that the average GTP allocation level of China's resource-based cities showed a steady upward trend in 2009 to 2018, from 0.4196 in 2009 to 0.4946 in 2018, an increase of 17.88%, and the growth rate showed a "W"-type fluctuating change characteristics. (2) Spatial differentiation characteristics analysis results showed that the average GTP allocation level of resource-based cities showed the heterogeneous characteristics of "east>middle>west", "regenerative type> growth type> mature type> declining type", and "ferrous metals> oil and gas> non-metals> forest industry> coal> non-ferrous metals". (3) Overall policy effects analysis results showed that the implementation of the SDP policy had a weak positive promotion effect on the overall transformation and development of resource-based cities. The overall policy effect showed an "M"-type fluctuation trend, with a more obvious pushing effect at the early stage of policy implementation, and then showed a fluctuation and weakening trend. (4) Local policy effects analysis results showed that the implementation of the SDP policy had different impacts on the green transformation and development of different resource-based cities. This policy has positively promoted the GTP of mature cities (e.g., Panzhihua City) and declining cities (e.g., Jiaozuo City), while it has not significantly promoted the GTP of growth cities and regenerative cities. These findings can provide a reference for the optimization and improvement of sustainable development policies in resource-based cities in China.

Fluoride-Controlled Riboswitch-Based Dampening of Gene Expression for Cloning Potent Promoters.

Bioreporter systems based on detectable enzyme activity, such as that of beta-galactosidase or luciferase, are key in novel bacterial promoter discovery and study. While these systems permit quantification of gene expression, their use is limited by the toxicity of the expressed reporter enzymes in a given host. Indeed, the most potent promoters may be overlooked if their activity causes a lethal overproduction of the reporter genes when screening for transcriptional activity of potential promoter sequences with the luxCDABE cassette. To overcome this limitation, a variation of the mini-CTX-lux plasmid has been designed which allows reduction of promoter activity via the addition of an adjacent fluoride riboswitch. The riboswitch adds a layer of regulation between the promoter and the reporter gene, allowing cloning of stronger promoters by weakening expression, while giving the potential to induce with fluoride to provide a good signal for weaker promoters, thus circumventing limitations associated with reporter toxicity. We noticed the riboswitch potential portability issues between species, suggesting caution when using riboswitches non-native to the species where it is being used. This study introduces a new molecular biology tool which will allow for the identification of previously unverifiable or uncharacterized potent promoters and also provides a cloning vector for translational fusion with luciferase in a plasmid compatible with many species such as from the genera Burkholderia and Pseudomonas.

IPro-GAN: A novel model based on generative adversarial learning for identifying promoters and their strength

Background and ObjectivePromoter is a component of the gene, which can specifically bind with RNA polymerase and determine where transcription starts, and also determine the transcription efficiency of the gene. Promoters can be divided into strong promoters and weak promoters because their structures and the interaction time interval are quite different. The functional variation of the promoter can lead to a variety of diseases. Therefore, identifying promoters and their strength is necessary and has important biological significance. A novel and promising model based on deep learning is proposed to achieve it. MethodsIn this work, we build a power model named iPro-GAN for identification of promoters and their strength. First, we collect benchmark datasets and independent datasets for training and testing. Then, Moran-based spatial auto-cross correlation method is used as feature extraction method. Finally, deep convolution generative adversarial network with 10-fold cross validation is applied for classifying. The first layer of the model is used to identify the promoter and the second layer is used to determine its type. ResultsOn the benchmark data set, the accuracy of the first layer predictor is 93.15%, and the accuracy of the second layer predictor is 92.30%. On the independent data set, the accuracy of the first layer predictor is 86.77%, and the accuracy of the second layer predictor is 91.66%. In particular, breakthrough progress has been made in the identification of promoters’ strength. ConclusionsThese results are far higher than the existing best predictor, which indicate that our model is serviceable and practicable to identify promoters and their strength. Furthermore, the datasets and source codes are available from this link: https://github.com/Bovbene/iPro-GAN.

Increased expression of the fluorescent reporter protein ymNeonGreen in Saccharomyces cerevisiae by reducing RNA secondary structure near the start codon.

Expression of a new fluorescent reporter protein called mNeonGreen, that is not based on the jellyfish green fluorescent protein (GFP) sequence, shows increased brightness and folding speed compared to enhanced GFP. However, in vivo brightness of mNeonGreen and its yeast-optimized variant ymNeonGreen in S. cerevisiae is lower than expected, limiting the use of this high quantum yield, fast-folding reporter in budding yeast. This study shows that secondary RNA structure near the start codon in the ymNeonGreen ORF inhibits expression in S. cerevisiae. Removing secondary structure, without altering the ymNeonGreen protein sequence, led to a 2 and 4-fold increase in fluorescence when expressed in S. cerevisiae and E. coli, respectively. In S. cerevisiae, increased fluorescence was seen with strong and weak promoters and led to higher transcript levels suggesting greater transcript stability and improved expression in the absence of stable secondary RNA structure near the start codon.

Current State and Prospects of Development of Sports and Health Tourism in Bukovyna

Purpose. Assess the current state and prospects of sports and health tourism in Bukovyna. Methods.Field, surveys, statistics, SWOT-analysis of the state and development of tourism. Results. 156 tourist organizations operate in the region's tourist services market. Of the eleven districts of the region, six sports and health tourism is developing quite rapidly: Vyzhnytskyi, Kitsmanskyi, Novoselytskyi, Putylskyi, Storozhynetskyi and Khotynskyi. In the course of the research, a number of survey questions were developed and a survey of 365 respondents was conducted. Respondents included participants of all types of tourist activities in Bukovyna: tourists who are members of various tourist clubs, participants of spontaneous campaigns, members of spontaneous groups at youth organizations, educational institutions. Employees of the regional tourism department, as well as heads of tourist clubs, their deputies, presidents and members of the presidium of the Chernivtsi regional and city sports tourism federations also took part in the survey. The statistical analysis of the results of the survey on the proposed system of questions showed that respondents agree on the complexity of the situation with the financing and logistics of tourism, including sports and health. The imperfection of the structure and content of professional training, the imperfection of the program, regulatory and legal framework, as well as insufficient attention to this type of tourism by the state and weak promotion. Conclusions. The availability of natural resources in the region, the demand for recreation determine the interest in the widespread development of sports and health tourism in the region, provided the creation of a mechanism to stimulate investment in tourism infrastructure and the appropriate level of information technology.

Evaluation of inducible promoter-riboswitch constructs for heterologous protein expression in the cyanobacterial species Anabaena sp. PCC 7120.

Cyanobacteria are promising chassis for synthetic biology applications due to the fact that they are photosynthetic organisms capable of growing in simple, inexpensive media. Given their slower growth rate than other model organisms such as Escherichia coli and Saccharomyces cerevisiae, there are fewer synthetic biology tools and promoters available for use in model cyanobacteria. Here, we compared a small library of promoter–riboswitch constructs for synthetic biology applications in Anabaena sp. PCC 7120, a model filamentous cyanobacterium. These constructs were designed from six cyanobacterial promoters of various strengths, each paired with one of two theophylline-responsive riboswitches. The promoter–riboswitch pairs were cloned upstream of a chloramphenicol acetyltransferase (cat) gene, and CAT activity was quantified using an in vitro assay. Addition of theophylline to cultures increased the CAT activity in almost all cases, allowing inducible protein production with natively constitutive promoters. We found that riboswitch F tended to have a lower induced and uninduced production compared to riboswitch E for the weak and medium promoters, although the difference was larger for the uninduced production, in accord with previous research. The strong promoters yielded a higher baseline CAT activity than medium strength and weak promoters. In addition, we observed no appreciable difference between CAT activity measured from strong promoters cultured in uninduced and induced conditions. The results of this study add to the genetic toolbox for cyanobacteria and allow future natural product and synthetic biology researchers to choose a construct that fits their needs.

Optimized Doxycycline-Inducible Gene Expression System for Genetic Programming of Tumor-Targeting Bacteria

PurposeIn the programming of tumor-targeting bacteria, various therapeutic or reporter genes are expressed by different gene-triggering strategies. Previously, we engineered pJL87 plasmid with an inducible bacterial drug delivery system that simultaneously co-expressed two genes for therapy and imaging by a bidirectional tet promoter system only in response to the administration of exogenous doxycycline (Doxy). In this multi-cassette expression approach, tetA promoter (PtetA) was 100-fold higher in expression strength than tetR promoter (PtetR). In the present study, we developed pJH18 plasmid with novel Doxy-inducible gene expression system based on a tet promoter.ProceduresIn this system, Tet repressor (TetR) expressed by a weak constitutive promoter binds to tetO operator, resulting in the tight repression of gene expressions by PtetA and PtetR, and Doxy releases TetR from tetO to de-repress PtetA and PtetR.ResultsIn Salmonella transformed with pJH18, the expression balance of bidirectional tet promoters in pJH18 was remarkably improved (PtetA:PtetR = 4~6:1) compared with that of pJL87 (PtetA:PtetR = 100:1) in the presence of Doxy. Also, the expression level by novel tet system was much higher in Salmonella transformed with pJH18 than in those with pJL87 (80-fold in rluc8 and 5-fold in clyA). Interestingly, pJH18 of the transformed Salmonella was much more stably maintained than pJL87 in antibiotic-free tumor-bearing mice (about 41-fold), because only pJH18 carries bom sequence with an essential role in preventing the plasmid-free population of programmed Salmonella from undergoing cell division.ConclusionsOverall, doxycycline-induced co-expression of two proteins at similar expression levels, we exploited bioluminescence reporter proteins with preclinical but no clinical utility. Future validation with clinically compatible reporter systems, for example, suitable for radionuclide imaging, is necessary to develop this system further towards potential clinical application.

IPromoter-ET: Identifying promoters and their strength by extremely randomized trees-based feature selection

Promoter is a region of DNA that determines the transcription of a particular gene. There are several σ factors in the RNA polymerase, which has the function of identifying the promoter and facilitating the binding of the RNA polymerase to the promoter. Owing to the importance of promoter in genome research, it is an urgent task to develop computational tool for effectively identifying promoters and their strength facing the avalanche of DNA sequences discovered in the post-genomic age. In this paper, we develop a model named iPromoter-ET using the k-mer nucleotide composition, binary encoding and dinucleotide property matrix-based distance transformation for features extraction, and extremely randomized trees (extra trees) for feature selection. Its 1st layer is used to identify whether a DNA sequence is of promoter or not, while its 2nd layer is to identify promoter samples as being strong or weak promoter. Support vector machine and the five cross-validation are used to perform identification and assess performance, respectively. The results indicate that our model remarkably outperforms the existing models in both the 1st and 2nd layers for accuracy and stability. We anticipate that our proposed model will become a very effective intelligent tool, or at the least, a complementary tool to the existing modes of identifying promoters and their strength. Moreover, the datasets and codes for iPromoter-ET are freely available at https://github.com/shengli0201/iPromoter-ET.

A CRISPR/Cas9-based single-stranded DNA recombineering system for genome editing of Rhodococcus opacus PD630

Genome engineering of Rhodococcus opacus PD630, an important microorganism used for the bioconversion of lignin, is currently dependent on inefficient homologous recombination. Although a CRISPR interference procedure for gene repression has previously been developed for R. opacus PD630, a CRISPR/Cas9 system for gene knockout has yet to be reported for the strain. In this study, we found that the cytotoxicity of Cas9 and the deficiency in pathways for repairing DNA double-strand breaks (DSBs) were the major causes of the failure of conventional CRISPR/Cas9 technologies in R. opacus, even when augmented with the recombinases Che9c60 and Che9c61. We successfully developed an efficient single-stranded DNA (ssDNA) recombineering system coupled with CRISPR/Cas9 counter-selection, which facilitated rapid and scarless editing of the R. opacus genome. A two-plasmid system, comprising Cas9 driven by a weak Rhodococcus promoter Pniami, designed to prevent cytotoxicity, and a single-guide RNA (sgRNA) under the control of a strong constitutive promoter, was proven to be appropriate with respect to cleavage function. A novel recombinase, RrRecT derived from a Rhodococcus ruber prophage, was identified for the first time, which facilitated recombination of short ssDNA donors (40–80 nt) targeted to the lagging strand and enabled us to obtain a recombination efficiency up to 103-fold higher than that of endogenous pathways. Finally, by incorporating RrRecT and Cas9 into a single plasmid and then co-transforming cells with sgRNA plasmids and short ssDNA donors, we efficiently achieved gene disruption and base mutation in R. opacus, with editing efficiencies ranging from 22 % to 100 %. Simultaneous disruption of double genes was also confirmed, although at a lower efficiency. This effective genome editing tool will accelerate the engineering of R. opacus metabolism.

Design and development of Machinga mobile trading application: A participatory and design science research

In Tanzania, street traders face the challenge of limited markets caused by employing weak marketing and promotion strategies. This study developed a mobile application to solve the problem addressed using participatory design. Qualitative data were collected using focus group discussions and brainstorming from 80 respondents involving both street traders and customers in different workshops and meetings. Data were used for the design and development of the Machinga application. Furthermore, quantitative data for application evaluation were collected from 96 respondents using questionnaires. In addition, 20 interviews were conducted to validate the evaluation results. Thematic and descriptive analysis were performed for both qualitative and quantitative data. The results show that the mobile application has prospective features which solve the problem of limited markets in the street trading community. The application is perceived positively by end-users because of embracing their prior requirements and meeting the evaluation criteria of usefulness, ease-of-use, learnability, and user satisfaction. The study recommends further training of users to enable the application to attain its multiplier effect on the vast population. This study confirms the relevance of participatory design in ICT4D projects for informal workers as it allowed the involvement of end-users and reflected their voices in terms of the technology they desire.

A Study on Attracting Local And International Investments and Improving an Integrated Logistics System Allows Local Products to Reach Global Markets

This paper studies how local non-oil Omani products could reach global markets by attracting local and international investors and improving the integrated logistics system in Oman. Therefore, the scope of this research is investment agencies in Oman, which are the Ministry of Commerce, Industry, and Investment Promotion and the Public Authority for Investment Promotion and Export Development. This study targets the leading logistics companies in Oman, such as Asyad Group. The study used an online survey and direct interview targeting specialists and experts in investment agencies and logistics companies. The results of the research showed that challenges are facing attracting local and international investors in local industries. Those challenges are the lengthy, complicated and costly procedures to obtain permits and licenses for investors, weak promotion of investment opportunities and local products at the international level, weakness of the local market, cumbersome regulations of the government, and so on. All these challenges constitute an obstacle to attracting investments. Findings also vindicate that weaknesses in logistical performance represented by the complex export and import procedures, complications in customs clearance operations, and the weak performance of land, sea, and air transport, which constitutes a barrier to exporting local products to global markets. The primary limitations that the researchers faced in conducting this research. First, there were many complications in collecting information through interviews. Some companies refused to conduct interviews due to preventive measures for the spread of the Coronavirus, which may affect the results.

Core promoter activity contributes to chromatin-based regulation of internal cryptic promoters.

During RNA polymerase II (RNA Pol II) transcription, the chromatin structure undergoes dynamic changes, including opening and closing of the nucleosome to enhance transcription elongation and fidelity. These changes are mediated by transcription elongation factors, including Spt6, the FACT complex, and the Set2-Rpd3S HDAC pathway. These factors not only contribute to RNA Pol II elongation, reset the repressive chromatin structures after RNA Pol II has passed, thereby inhibiting aberrant transcription initiation from the internal cryptic promoters within gene bodies. Notably, the internal cryptic promoters of infrequently transcribed genes are sensitive to such chromatin-based regulation but those of hyperactive genes are not. To determine why, the weak core promoters of genes that generate cryptic transcripts in cells lacking transcription elongation factors (e.g. STE11) were replaced with those from more active genes. Interestingly, as core promoter activity increased, activation of internal cryptic promoter dropped. This associated with loss of active histone modifications at the internal cryptic promoter. Moreover, environmental changes and transcription elongation factor mutations that downregulated the core promoters of highly active genes concomitantly increased their cryptic transcription. We therefore propose that the chromatin-based regulation of internal cryptic promoters is mediated by core promoter strength as well as transcription elongation factors.

COMPUTATIONAL IDENTIFICATION OF PROMOTER REGIONS IN PROKARYOTES AND EUKARYOTES

Promoters are modular DNA structures that contain complex regulatory elements required for the initiation of gene transcription. Therefore, the use of machine learning methods to identify promoters is very important for improving genome annotation and understanding transcriptional regulation. In recent years, many methods for predicting eukaryotic and prokaryotic promoters have been proposed. However, the performance of these methods is still far from satisfactory. In this article, we have developed a hybrid method (called IPMD) that combines a position correlation score function and diversity increment with modified Mahalanobis Discriminant to predict eukaryotic and prokaryotic promoters. The precise calculation and identification of promoters remains a challenge because these key DNA regulatory regions have variable structures composed of functional motifs that can provide gene-specific transcription initiation. The promoter is a regulatory DNA region, which is very important for gene transcription regulation. It is located near the transcription start site (TSS) upstream of the corresponding gene. In the post-genomics era, the availability of data makes it possible to build computational models to detect promoters robustly, because these models are expected to be helpful to academia and drug discovery. Until recently, the developed model only focused on distinguishing sequences into promoters and non-promoters. However, by considering the classification of weak and strong promoters, promoter predictors can be further improved. INDEX TERMS—: deep learning, DNA sequence analysis, Promoter prediction, Promoters, Promoter elements

Hybrid promoter engineering strategies in Yarrowia lipolytica: isoamyl alcohol production as a test study

BackgroundIn biological cells, promoters drive gene expression by specific binding of RNA polymerase. They determine the starting position, timing and level of gene expression. Therefore, rational fine-tuning of promoters to regulate the expression levels of target genes for optimizing biosynthetic pathways in metabolic engineering has recently become an active area of research.ResultsIn this study, we systematically detected and characterized the common promoter elements in the unconventional yeast Yarrowia lipolytica, and constructed an artificial hybrid promoter library that covers a wide range of promoter strength. The results indicate that the hybrid promoter strength can be fine-tuned by promoter elements, namely, upstream activation sequences (UAS), TATA box and core promoter. Notably, the UASs of Saccharomyces cerevisiae promoters were reported for the first time to be functionally transferred to Y. lipolytica. Subsequently, using the production of a versatile platform chemical isoamyl alcohol as a test study, the hybrid promoter library was applied to optimize the biosynthesis pathway expression in Y. lipolytica. By expressing the key pathway gene, ScARO10, with the promoter library, 1.1–30.3 folds increase in the isoamyl alcohol titer over that of the control strain Y. lipolytica Po1g KU70∆ was achieved. Interestingly, the highest titer increase was attained with a weak promoter PUAS1B4-EXPm to express ScARO10. These results suggest that our hybrid promoter library can be a powerful toolkit for identifying optimum promoters for expressing metabolic pathways in Y. lipolytica.ConclusionWe envision that this promoter engineering strategy and the rationally engineered promoters constructed in this study could also be extended to other non-model fungi for strain improvement.

Environmental Courts, Environment and Employment: Evidence from China

Whether environmental governance will cause unemployment has always been an aspect that the government needs to pay attention to in the process of making environmental policies, and is also a concern of residents. This paper analyzes the policy effect of environmental courts, which is a very important policy tool for the legalization of China’s environmental governance. While investigating whether environmental courts can effectively improve environmental quality, we also analyze its possible impact on employment and the specific mechanisms. The results show that: (1) After the establishment of environmental courts, the PM2.5 concentration has been significantly reduced. (2) While improving the environmental quality, environmental courts will produce a weak employment promotion effect. (3) Environmental courts affect the amount of employment through cost effect, factor substitution effect and innovation effect. This study provides empirical evidence for China and other developing countries to promote the legalization of environmental governance.

HbA2 induction: the merit of pancellularity in sickle cell disease.

HbA2 induction: the merit of pancellularity in sickle cell disease.