Strain Improvement Research Articles

Heterogeneous A40926 Self-Resistance Profile in Nonomuraea gerenzanensis Population Informs Strain Improvement

Nonomuraea gerenzanensis ATCC 39727 produces the glycopeptide antibiotic A40926, which is the natural precursor of the semi-synthetic, last-resort drug dalbavancin. To reduce the cost of dalbavancin production, it is mandatory to improve the productivity of the producing strain. Here, we report that the exposure of N. gerenzanensis wild-type population to sub-inhibitory concentrations of A40926 led to the isolation of differently resistant phenotypes to which a diverse A40926 productivity was associated. The most resistant population (G, grand colonies) represented at least the 20% of the colonies growing on 2 µg/mL of A40926. It showed a stable phenotype after sub-culturing and a homogeneous profile of self-resistance to A40926 in population analysis profile (PAP) experiments. The less resistant population (P, petit) was represented by slow-growing colonies to which a lower A40926 productivity was associated. At bioreactor scale, the G variant produced twice more than the wild-type (ca. 400 mg/L A40926 versus less than 200 mg/L, respectively), paving the way for a rational strain improvement based on the selection of increasingly self-resistant colonies.

Understanding the Role of Trichoderma reesei Vib1 in Gene Expression during Cellulose Degradation.

Vib1, a member of the Ndt80/PhoG-like transcription factor family, has been shown to be essential for cellulase production of Trichoderma reesei. Here, we combined transcriptomic and genetic analyses to gain mechanistic insights into the roles of Vib1 during cellulose degradation. Our transcriptome analysis showed that the vib1 deletion caused 586 genes with decreased expression and 431 genes with increased expression on cellulose. The downregulated genes were enriched for Gene Ontology terms associated with carbohydrate metabolism, transmembrane transport, oxidoreductase activity, and transcription factor activity. Of the 258 genes induced by cellulose, 229 showed no or decreased expression in Δvib1 on cellulose, including almost all (hemi)cellulase genes, crucial sugar transporter genes (IDs:69957, 3405), and the genes encoding main transcriptional activators Xyr1 and Ace3. Additionally, Vib1 also regulated the expression of genes involved in secondary metabolism. Further comparison of the transcriptomes of Δvib1 and Δxyr1 in cellulose revealed that the genes regulated by Vib1 had much overlap with Xyr1 targets especially for the gene set induced by cellulose, presumably whose expression requires the cooperativity between Vib1 and Xyr1. Genetic evidence indicated that Vib1 regulates cellulase gene expression partially via Xyr1. Our results will provide new clues for strain improvement.

A putative redox-sensing regulator Rex regulates lincomycin biosynthesis in Streptomyces lincolnensis.

Lincomycin is an important antimicrobial agent which is widely used in clinical and animal husbandry. The biosynthetic pathway of lincomycin comes to light in the past 10 years, however, the regulatory mechanism is still unclear. In this study, a redox-sensing regulator Rex from Streptomyces lincolnensis (Rexlin ) was identified and characterized to affect cell growth and lincomycin biosynthesis. Disruption of rex resulted in an increase in cell growth, but a decrease in lincomycin production. The results of quantitative real-time polymerase chain reaction showed that Rexlin can promote transcription of the regulatory gene lmbU and the structural genes lmbA, lmbC, lmbJ, lmbV, and lmbW. However, electrophoretic mobility shift assay analysis demonstrated that Rexlin can not bind to the promoter regions of these genes above. Findings in this study broadened our horizons in the regulatory mechanism of lincomycin production and laid a foundation for strain improvement of antibiotic producers.

Harnessing the yeast Saccharomyces cerevisiae for the production of fungal secondary metabolites.

Fungal secondary metabolites (FSMs) represent a remarkable array of bioactive compounds, with potential applications as pharmaceuticals, nutraceuticals, and agrochemicals. However, these molecules are typically produced only in limited amounts by their native hosts. The native organisms may also be difficult to cultivate and genetically engineer, and some can produce undesirable toxic side-products. Alternatively, recombinant production of fungal bioactives can be engineered into industrial cell factories, such as aspergilli or yeasts, which are well amenable for large-scale manufacturing in submerged fermentations. In this review, we summarize the development of baker’s yeast Saccharomyces cerevisiae to produce compounds derived from filamentous fungi and mushrooms. These compounds mainly include polyketides, terpenoids, and amino acid derivatives. We also describe how native biosynthetic pathways can be combined or expanded to produce novel derivatives and new-to-nature compounds. We describe some new approaches for cell factory engineering, such as genome-scale engineering, biosensor-based high-throughput screening, and machine learning, and how these tools have been applied for S. cerevisiae strain improvement. Finally, we prospect the challenges and solutions in further development of yeast cell factories to more efficiently produce FSMs.

Sweet sorghum juice as an alternative carbon source and adaptive evolution of Lactobacillus brevis NIE9.3.3 in sweet sorghum juice and biodiesel derived crude glycerol to improve 1, 3 propanediol production

Sweet sorghum juice (SSJ) is considered as an ideal complement for carbon supplement in ethanol fermentation for its ease of cultivation. Extraction of fermentable sugars from the sweet sorghum is very simple in comparison to lignocellulosic biomass. Hence sweet sorghum is a suitable candidate as a feedstock. In the present study, batch fermentations were carried out using Lactobacillus brevis NIE9.3.3, a facultative anaerobe, isolated through onsite enrichment technique to produce 1,3-propanediol and other co-metabolites, in glucose-glycerol co-fermentation. To make the process more sustainable, the glucose supplemented in the production media was replaced with SSJ. The supplementation of 40 g/L sorghum juice and 40 g/L crude glycerol in the production media resulted in the titre of 25.9 g/L 1, 3-PDO with a volumetric yield of 0.64 g 1,3-PDO/g glycerol. Adaptation of the microorganisms and cultivation under controlled conditions of temperature and substrate concentrations followed by selection was carried out, that is, adaptive evolution. Among the adaptively evolved strains, PD 20.100 has displayed better performance and increased the titres up to 38.4 g/L with a volumetric yield of 0.64 g 1,3-PDO/g glycerol. The industrial applicability of the fermentation process was checked in pilot scale and the production yield was comparable with that of flask scale. The utilization of agricultural and biodiesel industrial waste for the production of 1,3-PDO by a non-pathogenic organism and the strain improvement through ALE for better utilization and conversion of substrates indicates the novelty of this work.

Experimental Method for Tensile Testing of Unidirectional Carbon Fibre Composites Using Improved Specimen Type and Data Analysis.

This paper presents an experimental method for tensile testing of unidirectional carbon fibre composites. It uses a novel combination of a new specimen geometry, protective layer, and a robust data analysis method. The experiments were designed to test and analyze unprotected (with conventional end-tabs) and protected (with continuous end-tabs) carbon fibre composite specimens with three different specimen geometries (straight-sided, butterfly, and X-butterfly). Initial stiffness and strain to failure were determined from second-order polynomial fitted stress–strain curves. A good agreement between back-calculated and measured stress–strain curves is found, on both composite and fibre level. For unprotected carbon composites, the effect of changing specimen geometry from straight-sided to X-butterfly was an increase in strain to failure from 1.31 to 1.44%. The effect of protection on X-butterfly specimens was an increase in strain to failure from 1.44 to 1.53%. For protected X-butterfly specimens, the combined effect of geometry and protection led to a significant improvement in strain to failure of 17% compared to unprotected straight-sided specimens. The observed increasing trend in the measured strain to failure, by changing specimen geometry and protection, suggests that the actual strain to failure of unidirectional carbon composites is getting closer to be realized.

Unilateral branch pulmonary artery stenting in tetralogy of fallot improves ventricular function

Abstract Funding Acknowledgements Type of funding sources: Public grant(s) – National budget only. Main funding source(s): NIHR Background Pulmonary artery stenosis (PAS) occurs commonly in patients with tetralogy of fallot (ToF). Cardiac function and especially left ventricular longitudinal strain has been identified as an important prognostic factor for long term survival in ToF. The clinical relevance of unilateral PAS to long-term bi-ventricular function is poorly understood. Purpose We sought to evaluate the effect of resolving unilateral pulmonary artery obstruction on right and left ventricular performance. Methods We prospectively included 40 patients with TOF between 2016 and 2020, 20 who underwent unilateral PAS stenting and as comparison 20 who underwent surgical pulmonary valve replacement (PVR). MRI data was acquired during routine clinical care before and around 6-12 months after the procedure. 4 PAS patients attended additional research scans acquiring ventricular 4D flow MRI data. 4D flow MRI data was compared to the average kinetic energy curve of 10 age-matched healthy volunteers. Results Of the 20 patients undergoing PAS, 2 also underwent percutaneous PVR and were excluded from the comparison analysis. All patients in the PAs group showed an improvement in branch PA flow differential post procedure. Patients undergoing PAS were younger than those undergoing PVR (median 12 vs 19 years, p < 0.001). Other baseline anatomical and functional parameters including right ventricular (RV) volume indexed to body surface are (RVEDV/BSA) were comparable (pre PAS median 151 [122,170] vs pre PVR 162 [140,191]; p = 0.217). While in the PVR group the right ventricular volumes reduced in both end-diastole and end-systole, in the PAS group RV function improved due to reduced end-systolic volume with largely stable end-diastolic volumes. Changes in the left ventricle (LV) were even more interesting. In the PVR group ejection fraction improved due to an increase in end-diastolic volume with no improvement in ventricular longitudinal strain. In contrast, in the PAS group LV ejection fraction improved by a reduction in end-systolic volume and the PAS group showed a small but significant improvement in LV longitudinal strain. In addition, ¾ patients undergoing 4D flow MRI assessment showed LV kinetic energy curve more similar to the healthy volunteer averaged LV kinetic energy curve after PAS. The 4th patient already had a near normal LV kinetic energy curve prior to PAS. Conclusion Unilateral PAS does not alter RV end-diastolic volumes but improves RV function. LV ejection fraction improvement is similar to that seen after PVR, but importantly PAS also improved LV longitudinal strain. This suggests that PAS might positively influence long term morbidity and mortality risk in ToF patients, but a larger multi-centre long term follow-up study is needed to confirm this.

Microbial Cellulases: A Review on Strain Development, Purification, Characterization and their Industrial Applications

In this advance era, the enzymes are considered as a core kernel of white biotechnology and their demand is increasing day by day. According to report published in Research and Markets (ID: 5009185), the estimated global market for industrial enzymes were USD 10.0 billion in 2019, which is continuously increasing as it is expected to reach about USD 14.7 billion by 2022. Among all enzymes, cellulases are the major group of enzymes act synergistically in breakdown of cellulose, that facilitates its conversion to various value-added products and also offer several other important applications at industrial scale. The hyper production of cellulases are required to overcome their demand of global market. Cellulases production can be enhanced by strain improvement as well as using advance fermentation technology. In this review a detail studies of strategies to enhance production of cellulases and improve their physiochemical properties for industrial application have been described.

Reliable self-healing FBG sensor network for improvement of multipoint strain sensing

In this paper, we proposed and designed reliable self-healing fiber Bragg grating (FBG) sensor network for improvement of multipoint strain sensing. We use an optical switch (OSW) to reconfigure the sensor network and improve the self-healing function during fault happen in the sensor network. In this work, to prove and validate the detection performance of our proposed Bragg wavelength detection technique even when the number of overlap spectra of FBGs increases, we conduct three experiments and collect three different cases of strain data (namely case 1, case 2, and case 3) by applying different strain steps to FBGs. In case 1 experiment, strain was applied to only FBG1 sensor, while the other four FBGs keeps fixed. In case 2 experiment, strain was applied to FBG1 and FBG2 sensors at the same time and with different strain steps. In case 3 experiment, strain was applied to FBG1, FBG2 and FBG3 sensors at the same time and with different strain steps. As a result, in all cases of the experiment, three situations of spectra were introduced between FBGs like non-overlapped, partially overlapped and completely overlapped spectra. To solve this overlap problem, we used deep learning technique to accurately identify the Bragg wavelength of FBGs in the condition of the partially or fully overlapped spectra. Therefore, our proposed FBG sensor system can improve the reliability and detection accuracy of the sensor system even the number of overlaps FBGs spectra increases.

Effect of surgical treatment on myocardial strain in patients with pheochromocytoma and paraganglioma: a mini-review and meta-analysis.

Information on systolic dysfunction, as assessed by left-ventricular (LV) mechanics, in patients with pheochromocytoma after surgical treatment is scanty. We performed a systematic meta-analysis of speckle tracking echocardiographic studies to provide an updated comprehensive information on this issue. The PubMed, OVID-MEDLINE, and Cochrane library databases were analyzed to search for articles published from the inception up to May 31st 2021. Studies were identified using MeSH terms and crossing the following search items: "myocardial strain" "left ventricular mechanics", "speckle tracking echocardiography", "systolic dysfunction", "pheochromocytoma", and "paraganglioma". A total of 92 surgically treated patients with pheochromocytoma/paraganglioma were included in 3 longitudinal studies. Successful surgical treatment was associated with a decrease in relative wall thickness (SMD -0.25 ± 0.10, CI -0.45/-0.05, p < 0.01) and an improvement in global longitudinal strain (SMD -0.45 ± 0.10, CI -0.66/-0.24, p < 0.0001). The favorable effects of treatment on LV geometry and mechanics were not accompanied by significant changes in ejection fraction (SMD -0.07 ± 0.10, CI -0.27/0.12, p = 0.44). This meta-analysis adds a new piece of evidence, suggesting that surgical treatment of patients with pheochromocytoma impacts favorably on LV geometry and LV mechanics, and, more importantly, the assessment of LV changes in this setting can no longer rely on conventional echocardiographic parameters such as ejection fraction.

Genetics and metabolic engineering of yeast strains for efficient ethanol production

AbstractBioethanol production from monomeric sugar is performed by several yeasts. But there are several limitations associated with yeast strains such as their low tolerance to ethanol, toxic inhibitors, and high sugar concentration. Genetic and metabolic engineering of potential yeast strains can overcome the above limitations. The present article summarized current genetic and metabolic engineering approaches for the development of yeast strain for efficient ethanol production. The review systematically examined bioethanol generations based on substrate utilization, criteria for strain selections, strategies for strain improvements including randomized mutagenesis, genetic engineering, metabolic engineering, genome editing, whole genome (re)sequencing, promoter engineering, quantitative trait locus analysis, protein engineering, and evolutionary engineering. Different fermentation technologies employed in hydrolysate fermentation including low gravity (LG), high gravity (HG), and very high gravity (VHG) as well as challenges of yeast strains development and its future prospect have been critically evaluated in this article. Significant engineering efforts are imminent for yeast‐based second‐generation biofuel to leave a demonstration phase through strain improvement and become economically competitive with fossil fuel.Practical ApplicationsThis is a comprehensive review of yeast strain development for bioethanol production. The readers should be able to acquire some basic knowledge on: The accompanied substrates for bioethanol generations as well as the technologies and challenges behind them. The criteria to consider in selecting yeast strain for bioengineering development. Different strategies and their reported applications employed in yeast strain development including randomized mutagenesis, genetic engineering, metabolic engineering, genome editing, whole genome (re)sequencing, promoter engineering, quantitative trait locus (QTL) analysis, protein engineering, and evolutionary engineering. Challenges and merits of different fermentation technologies employed in hydrolysate fermentation including LG, HG, and VHG. Possible challenges to encounter in developing yeast strain for bioethanol production. Desirable traits to consider in the selection and development of yeast strains for bioethanol production.

Discovery of the anticancer drug vinblastine from the endophytic Alternaria alternata and yield improvement by gamma irradiation mutagenesis.

Several fungal endophytes were isolated from some medicinal plants and screened for their ability to produce the anticancer drug vinblastine. An isolate was found to produce vinblastine (205·38 μg l-1 ), and the identity of the fungal vinblastine was confirmed by UV spectroscopic, high-performance liquid chromatography and electrospray ionization mass spectrometry analyses. Based on both morphological and molecular studies, the vinblastine-producing strain was identified as Alternaria alternata. Cytotoxic activities of the fungal vinblastine were evaluated against CHO-K1, MCF-7 and HepG-2 cell lines by the MTT assay. The proliferation of these cell lines was inhibited after treatment with fungal vinblastine and the recorded IC50 values of the respective cell lines were 12·15, 8·55 and 7·48 μg ml-1 . A strain improvement programme for improving vinblastine productivity by the fungal strain was also used. In addition, 10 broth media were evaluated for further increasing the production of vinblastine. The yield of vinblastine was intensified by 3·98-fold following gamma irradiation at 1000 Gy, and a stable mutant strain was isolated. Among the screened media, M1D broth (pH 6·0) stimulated the highest vinblastine production of 1553·62 μg l-1 by the isolated mutant strain. The present study is the first report on the production and yield improvement of the anticancer drug vinblastine by A. alternata. These findings suggest A. alternata as a viable and potent source with excellent biotechnological potential for the production of vinblastine.

Comparative transcriptome analysis of Trichoderma reesei reveals different gene regulatory networks induced by synthetic mixtures of glucose and \u03b2-disaccharide

The mixture of glucose and β-disaccharide (MGD) synthesized by transglycosylation of glucose as a low-cost soluble carbon source can efficiently induce cellulase production in Trichoderma reesei, which holds potential for the biorefining of lignocellulosic biomass. However, it is not yet fully understood how MGD induces T. reesei cellulase. In this study, transcriptomic analyses were conducted to investigate the molecular basis of MGD for lignocellulose-degrading enzyme production of T. reesei Rut C30 compared with that on lactose. Particular attention was paid to CAZymes, transcription factors, transporters and other protein processing pathways related to lignocellulose degradation. As a result, MGD can elicit transcription of GH5-, GH6- and GH7-encoding cellulases that is up to 1.4-fold higher than that induced by lactose, but GH11- and GH74-encoding xylanases are downregulated by 1.7- and 4.4-fold, respectively. Gene expression profiles suggest that the transcription activators xyr1 and vib1 are significantly upregulated and that the mitogen-activated protein kinase pathway is strengthened compared to the case of lactose induction. In addition, hac1-encoding UPR-specific transcription factors are significantly upregulated by MGD, which may be enhanced due to proper folding and processing of nascent proteins. These findings provide a theoretical basis for further understanding the characterization of efficient cellulase production using MGD as an inducer in T. reesei and offer potential strategies for strain improvement.

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.

Associations between physical activity, sedentary behaviour and left ventricular structure and function from the Echocardiographic Study of Latinos (ECHO-SOL)

ObjectiveThe cross-sectional association between accelerometer-measured physical activity (PA), sedentary behaviour (SB) and cardiac structure and function is less well described. This study’s primary aim was to compare echocardiographic measures of...

Factors Predicting Improvements in Global Longitudinal Strain After Native Valve and Valve-in-valve Transcatheter Aortic Valve Replacement With and Without Bioprosthetic Valve Fracture

Factors Predicting Improvements in Global Longitudinal Strain After Native Valve and Valve-in-valve Transcatheter Aortic Valve Replacement With and Without Bioprosthetic Valve Fracture

BIOCHEMICAL RESPONSES AND GENETIC EXPRESSIONS OF SYNTHETIC COMMON CARP POPULATIONS EXPOSED TO HIGH-AMMONIA REARING ENVIRONMENT

Carp is one of the leading freshwater aquaculture commodities in Indonesia. Further improvement of carp strains by the Research Institute for Fish Breeding (RIFB), Sukamandi, Indonesia, has produced a synthetic F2 carp. The strain is assembled from different strains of carps and has shown better growth and health characteristics. Considering that high environmental ammonia (HEA) has affected most carp grow-out systems, this study aimed to determine the performance of the synthetic carp populations in a high ammonia rearing environment. The treatments were rearing media of the synthetic carp seed populations added with and without (control) 200 mg/L NH4Cl arranged in three replicates. A total of 30 fish seeds/aquarium, weighed 10-15 g/fish, was used in the study. Dissolved oxygen levels were maintained above 2 mg/L using aeration. This study shows that higher tolerant carp populations had red blood cells of 232.66 ± 17.24 cells/mL, indicating a direct effect of high ammonia on red blood cell count (p&lt;0.05). Cortisol levels of 80.90 ± 6.35 ng/mL in resistant carp indicate significant differences (p&lt;0.05). The relative expressions of the HSP70 gene in the liver (Log10) ranged between 0.72 and 2.80. The values demonstrate that high ammonia-resistant synthetic carp have a higher relative expression ratio of the HSP70 gene than the less resistant group. This research concluded that the populations of synthetic F2 carp showed a degree of resistance against high-ammonia rearing conditions. When it is ready for aquaculture, this synthetic carp strain could be farmed in high density using intensive systems in HEA-affected artificial lakes and reservoir

Enhanced production of extracellular lipase by ethyl methane sulfonate from soil fungal strain in submerged and solid-state fermentation condition

Enhancement in the production of enzyme by utilizing different strains of microbe is one of the main prospects in biotechnology. In the present work, ethyl methane sulfonate (EMF) was selected as the chemical mutagen for inducing mutagenesis in fungi. It is a cheap method to induce random mutation as compared to other methods of recombinant technologies. Strain improvement was done by incubating the fungal spore suspension at variable concentrations of EMS i.e. 4% (v/v) and 10% (v/v) for the time period of 60, 90, and 120 min respectively. The set of control was treated with distilled water only. The fungal colonies were found to be maximum in control plate as compared to the EMF exposed plates. The number of fungal colonies was reduced as we raised the exposure time of EMF. Specific activity and the lipase activity of wild strain and hyperproducer were evaluated under the submerged (SmF) and solid-state fermentation (SSF). The wild strain denoted the 3.2 U/ml/min of enzymatic activity under SmF and 15.87 U/g/min of activity under SSF. In contrast, the best enzymatic activity was represented by S2St1 at 10% of EMS after the time period of 60 min i.e. 11.7 U/ml/min under SmF and 99.35 U/g/min under SSF after the time period of 72 hrs. Statistical analysis by using one-way ANOVA determined that the value of F calculated was lower than the F tabulated. So, there was a significant relation between the EMS percentage and time of exposure among the mutated strains. In conclusion, this soil fungal strain can be utilized to produce lipase enzyme for numerous industrial applications.

In silico analysis of promoter region and regulatory elements of mitogenome co-expressed trn gene clusters encoding for bio-pesticide in entomopathogenic fungus, Metarhizium anisopliae: strain ME1

BackgroundPest control strategies almost entirely rely on chemical insecticides, which cause environmental problems such as biosphere deterioration and emergence of resistant pests. Bio-pesticide is an alternative approach, which uses organisms such as entomopathogenic fungi, Metarhizium anisopliae, to control pests. Screening such potential organism at a molecular level and understanding their gene regulation mechanism is an important approach to reduce emergence of pesticide resistance and worsening of the biosphere. Understanding promoter regions which play a pivotal role in gene regulation is crucial. In particular, identification of the promoter regions in M. anisopliae Strain ME1 remains poorly understood. To our knowledge, the mitogenome trn gene clusters of M. anisopliae Strain ME1 were not characterized. Here, we used machine learning approach to identify and characterize the promoter regions, regulatory elements, and CpG island densities of 15 protein coding genes of entomopathogenic fungi, M. anisolpliae Strain ME1. ResultsThe current analysis revealed multiple transcription start sites (TSS) for all utilized sequences, except for promoter region genes of Pro-cob and Pro-nad5. With reference to the start codon (ATG), 85.3% of TSS was located above – 500 bp. Based on the standard predictive score at cut off value of 0.8a, the current study revealed 54.7% of predictive score greater than or equal from 0.9 promoter prediction score. Expectation maximization algorithm output identified five candidate motifs. Nonetheless, of all candidate motifs, MtrnI was revealed as the common promoter region motif with a value of 76.9% both in terms of size of binding sites and with an E value of 9.1E−054. Accordingly, we perceived that MtrnI serve as the binding site for tryptophan cluster with P value 0.0044 and C4 type zinc fingers functions as the binding site to regulate gene expression of M. anisopliae Strain ME1. The analysis revealed that mitogenome trn gene clusters of M. anisopliae Strain ME1 showed homologues evolutionary ancestor supported with a bootstrap value of 100%. ConclusionIdentified common candidate motifs and binding transcription factors through in silico approach are likely expected to contribute for better understanding of gene expression and strain improvement of M. anisopliae Strain ME1 for its bio-pesticides role.

Comparative transcriptome analysis reveals the underlying mechanism for over-accumulation of menaquinone-7 in Bacillus subtilis natto mutant

This study combined whole-genome sequencing, compound mutagenesis, and comparative transcriptome analysis methods to investigate the menaquinone-7 (MK-7) metabolism and regulation in Bacillus subtilis natto. Whole genome sequencing identified that the wild-type MK-7 producing strain BN0 contains one single circular chromosome in size of 4,119,230 bp, harboring 4603 protein-coding genes, and two plasmids in size of 64,309 and 5820 bp respectively. In addition, UV-ARTP (Ultraviolet -atmospheric and room temperature plasm) compound mutagenesis was performed in BN0, resulting in 50.6% improvement of MK-7 production in the mutant BN19-A. Further comparative transcriptome analysis revealed that: (1) the key genes (e.g glpA, dxs, ispF and ispH) involved in the glycerol metabolism pathway and MEP pathway were significantly up-regulated; (2) most genes related to the formation of spore were down-regulated; and (3) many genes (e.g tatA, bmr and bcr) involved in Tat secretion pathway, ABC transporters and MFS transporters were up-regulated. These results help us get insights into the underlying metabolic mechanism of MK-7 production in B. subtilis natto, and provide more potential molecular targets to be engineered for further strain improvement.