Strong Promoter Research Articles

Bioactive, full-length parathyroid hormone delivered using an adeno-associated viral vector.

Delivering the parathyroid hormone (PTH) gene has been attempted preclinically in a handful of studies, but delivering full-length PTH (1-84) using adeno-associated viral (AAV) vectors has not. Given the difficulty in achieving therapeutic levels of secreted proteins using gene therapy, this study seeks to determine the feasibility of doing so with PTH. An AAV vector was used to deliver human PTH driven by a strong promoter. We demonstrate the ability to secrete full-length PTH from various cell types in vitro. PTH secretion from hepatocytes was measured over time and a fluorescent marker was used to compare the secretion rate of PTH in various cell types. Potency was measured by the ability of PTH to act on the PTH receptors of osteosarcoma cells and induced proliferation. PTH showed potency in vitro by inducing proliferation in two osteosarcoma cell lines. In vivo, AAV was administered systemically in immunocompromised mice which received xenografts of osteosarcoma cells. Animals that received the highest dose of AAV-PTH had higher liver and plasma concentrations of PTH. All dosing groups achieved measurable plasma concentrations of human PTH that were above the normal range. The high-dose group also had significantly larger tumors compared to control groups on the final day of the study. The tumors also showed dose-dependent differences in morphology. When looking at endocrine signaling and endogenous bone turnover, we observed a significant difference in tibial growth plate width in animals that received the high-dose AAV as well as dose-dependent changes in blood biomarkers related to PTH. This proof-of-concept study shows promise for further exploration of an AAV gene therapy to deliver full-length PTH for hypoparathyroidism. Additional investigation will determine efficacy in a disease model, but data shown establish bioactivity in well-established models of osteosarcoma.

Novel switchable ECF sigma factor transcription system for improving thaxtomin A production in Streptomyces

The application of the valuable natural product thaxtomin A, a potent bioherbicide from the potato scab pathogenic Streptomyces strains, has been greatly hindered by the low yields from its native producers. Here, we developed an orthogonal transcription system, leveraging extra-cytoplasmic function (ECF) sigma (σ) factor 17 (ECF17) and its cognate promoter Pecf17, to express the thaxtomin gene cluster and improve the production of thaxtomin A. The minimal Pecf17 promoter was determined, and a Pecf17 promoter library with a wide range of strengths was constructed. Furthermore, a cumate inducible system was developed for precise temporal control of the ECF17 transcription system in S. venezuelae ISP5230. Theoretically, the switchable ECF17 transcription system could reduce the unwanted influences from host and alleviate the burdens introduced by overexpression of heterologous genes. The yield of thaxtomin A was significantly improved to 202.1 ± 15.3 μ g/mL using the switchable ECF17 transcription system for heterologous expression of the thaxtomin gene cluster in S. venezuelae ISP5230. Besides, the applicability of this transcription system was also tested in Streptomyces albus J1074, and the titer of thaxtomin A was raised to as high as 239.3 ± 30.6 μg/mL. Therefore, the inducible ECF17 transcription system could serve as a complement of the generally used transcription systems based on strong native constitutive promoters and housekeeping σ factors for the heterologous expression of valuable products in diverse Streptomyces hosts.

Characterization of Catechol-1,2-Dioxygenase (Acdo1p) From Blastobotrys raffinosifermentans and Investigation of Its Role in the Catabolism of Aromatic Compounds.

Gallic acid, protocatechuic acid, catechol, and pyrogallol are only a few examples of industrially relevant aromatics. Today much attention is paid to the development of new microbial factories for the environmentally friendly biosynthesis of industrially relevant chemicals with renewable resources or organic pollutants as the starting material. The non–conventional yeast, Blastobotrys raffinosifermentans, possesses attractive properties for industrial bio-production processes such as thermo- and osmotolerance. An additional advantage is its broad substrate spectrum, with tannins at the forefront. The present study is dedicated to the characterization of catechol-1,2-dioxygenase (Acdo1p) and the analysis of its function in B. raffinosifermentans tannic acid catabolism. Acdo1p is a dimeric protein with higher affinity for catechol (KM = 0.004 ± 0.001 mM, kcat = 15.6 ± 0.4 s–1) than to pyrogallol (KM = 0.1 ± 0.02 mM, kcat = 10.6 ± 0.4 s–1). It is an intradiol dioxygenase and its reaction product with catechol as the substrate is cis,cis-muconic acid. B. raffinosifermentans G1212/YIC102-AYNI1-ACDO1-6H, which expresses the ACDO1 gene under the control of the strong nitrate-inducible AYNI1 promoter, achieved a maximum catechol-1,2-dioxygenase activity of 280.6 U/L and 26.9 U/g of dry cell weight in yeast grown in minimal medium with nitrate as the nitrogen source and 1.5% glucose as the carbon source. In the same medium with glucose as the carbon source, catechol-1,2-dioxygenase activity was not detected for the control strain G1212/YIC102 with ACDO1 expression under the regulation of its respective endogenous promoter. Gene expression analysis showed that ACDO1 is induced by gallic acid and protocatechuic acid. In contrast to the wild-type strain, the B. raffinosifermentans strain with a deletion of the ACDO1 gene was unable to grow on medium supplemented with gallic acid or protocatechuic acid as the sole carbon source. In summary, we propose that due to its substrate specificity, its thermal stability, and its ability to undergo long-term storage without significant loss of activity, B. raffinosifermentans catechol-1,2-dioxygenase (Acdo1p) is a promising enzyme candidate for industrial applications.

Transcriptome Analysis and SNP Identification Reveal That Heterologous Overexpression of Two Uncharacterized Genes Enhances the Tolerance of Magnaporthe oryzae to Manganese Toxicity.

ABSTRACTManganese is a crucial trace element that constitutes the cofactors of many enzymes. However, excessive Mn2+ can be toxic for both prokaryotes and eukaryotes. The mechanism of fungal genetics and metabolism in response to Mn2+ stress remains understudied, warranting further studies. Magnaporthe oryzae is well-established as the most destructive pathogen of rice. A field strain, YN2046, more sensitive to Mn2+ toxicity than other strains, was obtained from a previous study. Herein, we explored the genetic mechanisms of Mn2+ sensitivity in YN2046 through comparative transcriptomic analyses. We found that many genes previously reported to participate in Mn2+ stress were not regulated in YN2046. These non-responsive genes might cause Mn2+ sensitivity in YN2046. Weight gene correlation network analysis (WGCNA) was performed to characterize the expression profile in YN2046. Some overexpressed genes were only found in the Mn2+ tolerant isolate YN125. Among these, many single nucleotide polymorphism (SNP) were identified between YN125 and YN2046, which might disrupt the expression levels of Mn responsive genes. We cloned two uncharacterized genes, MGG_13347 and MGG_16609, from YN125 and transformed them to YN2046 with a strong promoter. Our results showed that the heterologous overexpression of two genes in YN2046 restored its sensitivity. Transcriptomic and biochemical analyses were performed to understand Mn tolerance mechanisms mediated by the two heterologous overexpressed genes. Our results showed that heterologous overexpression of these two genes activated downstream gene expression and metabolite production to restore M. oryzae sensitivity to Mn, implying that SNPs in responsive genes account for different phenotypes of the two strains under Mn stress.IMPORTANCE Heavy metals are used for fungicides as they target phytopathogen in multiple ways. Magnaporthe oryzae is the most destructive rice pathogen and is threatening global rice production. In the eukaryotes, the regulation mechanisms of Mn homeostasis often focus on the posttranslation, there were a few results about regulation at transcript level. The comparative transcriptome analysis showed that fewer genes were regulated in the Mn-sensitive strain. WGCNA and SNP analyses found that mutations in promoter and coding sequence regions might disrupt the expression of genes involved in Mn detoxification in the sensitive strain. We transferred two unannotated genes that were cloned from the Mn-tolerant strain into a sensitive strain with strong promoters, and the transformants exhibited an enhanced tolerance to Mn2+ toxicity. Transcriptome and biochemistry results indicated that heterologous overexpression of the two genes enhanced the tolerance to Mn toxicity by reactivation of downstream genes in M. oryzae.

Evaluation of Aspergillus niger Six Constitutive Strong Promoters by Fluorescent-Auxotrophic Selection Coupled with Flow Cytometry: A Case for Citric Acid Production.

Aspergillus niger is an important industrial workhorse for the biomanufacturing of organic acids, proteins, etc. Well-controlled genetic regulatory elements, including promoters, are vital for strain engineering, but available strong promoters for A. niger are limited. Herein, to efficiently assess promoters, we developed an accurate and intuitive fluorescent-auxotrophic selection workflow based on mCherry, pyrG, CRISPR/Cas9 system, and flow cytometry. With this workflow, we characterized six endogenous constitutive promoters in A. niger. The endogenous glyceraldehyde-3-phosphate dehydrogenase promoter PgpdAg showed a 2.28-fold increase in promoter activity compared with the most frequently used strong promoter PgpdAd from A. nidulans. Six predicted conserved motifs, including the gpdA-box, were verified to be essential for the PgpdAg activity. To demonstrate its application, the promoter PgpdAg was used for enhancing the expression of citrate exporter cexA in a citric acid-producing isolate D353.8. Compared with the cexA controlled by PgpdAd, the transcription level of the cexA gene driven by PgpdAg increased by 2.19-fold, which is consistent with the promoter activity assessment. Moreover, following cexA overexpression, several genes involved in carbohydrate transport and metabolism were synergically upregulated, resulting in up to a 2.48-fold increase in citric acid titer compared with that of the parent strain. This study provides an intuitive workflow to speed up the quantitative evaluation of A. niger promoters and strong constitutive promoters for fungal cell factory construction and strain engineering.

Manganese Uptake to Wheat Shoot Meristems Is Differentially Influenced by Arbuscular Mycorrhiza Fungal Communities Adapted to Acidic Soil

Soil acidity is a strong promoter of the bioavailability of Al, Fe, and Mn, whose concentrations can sometimes reach toxic levels for plants. In agricultural soils, the use of arbuscular mycorrhizal fungi (AMF) has shown a protective influence on wheat growth under Mn toxicity. The intact extraradical mycelium (ERM) promotes faster AMF colonization, leading to a higher wheat shoot weight, lower Mn uptake, and changes in antioxidant enzyme activity. Its effect on the uptake and distribution of plant nutrients according to the developmental stage of shoot organs has seldomly been analyzed. In the present study, Mn, Mg, Ca, and K were quantified by ICP–MS in leaves and apical meristems of wheat grown in soil with two different ERM consortia, developed from the native plants Lolium rigidum (LOL) and Ornithopus compressus (ORN). The ORN treatment induced the highest wheat shoot weights and the lowest Mn levels. In the leaves, no significant differences were detected for Mg, Ca, or K, but in the apical meristems, the ORN treatment slightly lowered the Ca concentration. The AMF associated with ORN was seen to enhance wheat weight partly by protecting the zones of active growth against high Mn levels in Mn toxic soils. The use of ORN in acidic soils with Mn toxicity provides a sustainable alternative and an efficient complement to current farming practices to lower the negative impacts of farming on the environment.

High-level production of wild-type and oxidation-resistant recombinant alpha-1-antitrypsin in glycoengineered CHO cells.

Alpha-1-antitrypsin (A1AT) is a serine protease inhibitor which blocks the activity of serum proteases including neutrophil elastase to protect the lungs. Its deficiency is known to increase the risk of pulmonary emphysema as well as chronic obstructive pulmonary disease. Currently, the only treatment for patients with A1AT deficiency is weekly injection of plasma-purified A1AT. There is still today no commercial source of therapeutic recombinant A1AT, likely due to significant differences in expression host-specific glycosylation profile and/or high costs associated with the huge therapeutic dose needed. Accordingly, we aimed to produce high levels of recombinant wild-type A1AT, as well as a mutated protein (mutein) version for increased oxidation resistance, with N-glycans analogous to human plasma-derived A1AT. To achieve this, we disrupted two endogenous glycosyltransferase genes controlling core α-1,6-fucosylation (Fut8) and α-2,3-sialylation (ST3Gal4) in CHO cells using CRISPR/Cas9 technology, followed by overexpression of human α-2,6-sialyltransferase (ST6Gal1) using a cumate-inducible expression system. Volumetric A1AT productivity obtained from stable CHO pools was 2.5- to 6.5-fold higher with the cumate-inducible CR5 promoter compared to five strong constitutive promoters. Using the CR5 promoter, glycoengineered stable CHO pools were able to produce over 2.1 and 2.8 g/L of wild-type and mutein forms of A1AT, respectively, with N-glycans analogous to the plasma-derived clinical product Prolastin-C. Supplementation of N-acetylmannosamine to the cell culture media during production increased the overall sialylation of A1AT as well as the proportion of bi-antennary and disialylated A2G2S2 N-glycans. These purified recombinant A1AT proteins showed in vitro inhibitory activity equivalent to Prolastin-C and substitution of methionine residues 351 and 358 with valines rendered A1AT significantly more resistant to oxidation. The recombinant A1AT mutein bearing an improved oxidation resistance described in this study could represent a viable biobetter drug, offering a safe and more stable alternative for augmentation therapy.

Improvement of acetyl-CoA supply and glucose utilization increases l-leucine production in Corynebacterium glutamicum.

l-Leucine is an important essential amino acid with multiple industrial applications, whose market requirements cannot be met because of the low productivity. In this study, a strain of Corynebacterium glutamicum with high l-leucine yield was constructed to enhance its acetyl-CoA supply and glucose utilization. One copy of leuA under the control of a strong promoter was incorporated into the C. glutamicum genome. Then, acetyl-CoA supply was increased by the integration of a terminator in front of gltA and by the heterogeneous overexpression of acetyl-CoA synthetase (ACS) and deacetylase (CobB) derived from Escherichia coli. Next, the transcriptional regulator sugR was deleted to enhance glucose uptake via a phosphotransferase-mediated route. In fed-batch fermentation performed in a 5-L reactor, l-leucine production of 40.11 ± 0.73g L-1 was achieved under the optimized conditions, with an l-leucine yield and productivity of 0.25g g-1 glucose and 0.59g L-1 h-1 , respectively. These results represent a significant improvement in the l-leucine production of C. glutamicum, indicating that the process possesses high potential for industrial application. These strategies can be also expanded to enable the production of other value-added biochemicals derived from the intermediates of central carbon metabolism.

Physiological lentiviral vectors for the generation of improved CAR-T cells

Anti-CD19 chimeric antigen receptor (CAR)-T cells have achieved impressive outcomes for the treatment of relapsed and refractory B-lineage neoplasms. However, important limitations still remain due to severe adverse events (i.e., cytokine release syndrome and neuroinflammation) and relapse of 40%–50% of the treated patients. Most CAR-T cells are generated using retroviral vectors with strong promoters that lead to high CAR expression levels, tonic signaling, premature exhaustion, and overstimulation, reducing efficacy and increasing side effects. Here, we show that lentiviral vectors (LVs) expressing the transgene through a WAS gene promoter (AW-LVs) closely mimic the T cell receptor (TCR)/CD3 expression kinetic upon stimulation. These AW-LVs can generate improved CAR-T cells as a consequence of their moderate and TCR-like expression profile. Compared with CAR-T cells generated with human elongation factor α (EF1α)-driven-LVs, AW-CAR-T cells exhibited lower tonic signaling, higher proportion of naive and stem cell memory T cells, less exhausted phenotype, and milder secretion of tumor necrosis factor alpha (TNF-α) and interferon (IFN)-ɣ after efficient destruction of CD19+ lymphoma cells, both in vitro and in vivo. Moreover, we also showed their improved efficiency using an in vitro CD19+ pancreatic tumor model. We finally demonstrated the feasibility of large-scale manufacturing of AW-CAR-T cells in guanosine monophosphate (GMP)-like conditions. Based on these data, we propose the use of AW-LVs for the generation of improved CAR-T products.

Institutional pressures and firms’ environmental management behavior: the moderating role of slack resources

Firms’ environmental management behavior is divided into two dimensions: substantive and symbolic. The effects of coercive pressure, mimetic pressure, and normative pressure on these two dimensions are explored based on institutional theory. The moderating role of slack resources in the above relationships is examined using a resource-based view. Data from 193 Chinese listed companies in the heavy-polluting industries from 2014 to 2018 are used to test the hypotheses. The results show the strongest facilitator of substantive environmental management behavior is coercive pressure, while the strongest promoter of symbolic environmental management behavior is mimetic pressure. Slack resources positively moderate institutional pressures and firms’ environmental management behavior relationships partially. The conclusions are significant to both environmental practitioners and managers.

Multiplexed Promoter Engineering for Improving Thaxtomin A Production in Heterologous Streptomyces Hosts.

Thaxtomin A is a potent bioherbicide in both organic and conventional agriculture; however, its low yield hinders its wide application. Here, we report the direct cloning and heterologous expression of the thaxtomin A gene cluster in three well-characterized Streptomyces hosts. Then, we present an efficient, markerless and multiplex large gene cluster editing method based on in vitro CRISPR/Cas9 digestion and yeast homologous recombination. With this method, we successfully engineered the thaxtomin A cluster by simultaneously replacing the native promoters of the txtED operon, txtABH operon and txtC gene with strong constitutive promoters, and the yield of thaxtomin A improved to 289.5 µg/mL in heterologous Streptomyces coelicolor M1154. To further optimize the biosynthetic pathway, we used constraint-based combinatorial design to build 27 refactored gene clusters by varying the promoter strength of every operon, and the highest titer of thaxtomin A production reached 504.6 μg/mL. Taken altogether, this work puts forward a multiplexed promoter engineering strategy to engineer secondary metabolism gene clusters for efficiently improving fermentation titers.

High-Level 5-Methyltetrahydrofolate Bioproduction in Bacillus subtilis by Combining Modular Engineering and Transcriptomics-Guided Global Metabolic Regulation.

5-Methyltetrahydrofolate (5-MTHF) is the predominant folate form in human plasma, which has been widely used as a nutraceutical. However, the microbial synthesis of 5-MTHF is currently inefficient, limiting green and sustainable 5-MTHF production. In this study, the Generally Regarded As Safe (GRAS) microorganism Bacillus subtilis was engineered as the 5-MTHF production host. Three precursor supply modules were first optimized by modular engineering for strengthening the supply of guanosine-5-triphosphate (GTP) and p-aminobenzoic acid (pABA). Next, the impact of genome-wide gene expression on 5-MTHF biosynthesis was evaluated using transcriptome analyses, which identified key genes for 5-MTHF production. The effects of potential genes on 5-MTHF synthesis were verified by observing the genes' up-regulated by strong promoter P566 and those down-regulated by inhibition through the clustered regularly interspaced short palindromic repeat interference (CRISPRi). Finally, a key gene for improved 5-MTHF biosynthesis, comGC, was integrated into the genome of modular engineered strain B89 for its overexpression and facilitating efficient 5-MTHF synthesis, reaching 3.41 ± 0.10 mg/L with a productivity of 0.21 mg/L/h, which was the highest level achieved by microbial synthesis. The engineered 5-MTHF-producing B. subtilis developed in this work lays the foundation of further enhancing 5-MTHF production by microbial fermentation, which can be used for isolation and purification of 5-MTHF as food and nutraceutical ingredients.

Tuning Geraniol Biosynthesis via a Novel Decane-Responsive Promoter in Candida glycerinogenes.

Geraniol is a rose-scented monoterpene with significant commercial and industrial value in medicine, condiments, cosmetics, and bioenergy. Here, we first targeted geraniol as a reporter metabolite and explored the suitability and potential of Candida glycerinogenes as a heterologous host for monoterpenoid production. Subsequently, dual-pathway engineering was employed to improve the production of geraniol with a geraniol titer of 858.4 mg/L. We then applied a synthetic hybrid promoter approach to develop a decane-responsive hybrid promoter based on the native promoter PGAP derived from C. glycerinogenes itself. The hybrid promoter was able to be induced by n-decane with 3.6 times higher transcriptional intensity than the natural promoter PGAP. In particular, the hybrid promoter effectively reduces the conflict between cell growth and product formation in the production of geraniol. Ultimately, 1194.6 mg/L geraniol was obtained at the shake flask level. The strong and tunable decane-responsive hybrid promoter developed in this study provides an important tool for fine regulation of toxic terpenoid production in cells.

MO759: Indoxyl-Sulfate Activation of the AhR-NFΚB Pathway Promotes IL-6 Secretion and Subsequent Osteogenic Transition and Calcification of Interstitial Cells of the Aortic Valve

Abstract BACKGROUND AND AIMS Calcific aortic valve disease (CAVD) results from fibro-calcific degeneration of the aortic valve leaflets, causing haemodynamic disorders and major cardiovascular complications. In chronic kidney disease (CKD) patients, this pathological remodelling is more frequent, appears earlier, progresses more rapidly and leads to a mortality rate three times higher than in the general population. The uremic toxin indoxyl-sulfate (IS) is a powerful predictor of cardiovascular mortality in CKD patients and a strong promoter of aortic calcification. However, its role in aortic valve leaflets mineralization has never been studied. This work aimed to evaluate whether IS can influence the mineralization of primary human valvular interstitial cells (hVICs) cultured in vitro. METHOD Primary hVICs were isolated from aortic tricuspid valves collected from patients with CAVD undergoing valve replacement surgery at the Amiens Picardie University Hospital. The concentrations of IS used in this study were selected according to the EUTOX registry: IS normal [Isn]: 0.5 µg/mL, IS uremic [Isu]: 37 µg/mL and IS maximum [Ismax]: 233 µg/mL. Human VICs mineralization were induced by exposure to an osteogenic medium containing 2.4 mmol/L calcium and 2.5 mmol/L phosphate (pro-calcific condition) and quantified by the o-cresolphthalein complexone method. The osteogenic transition of hVICs was assessed by qRT-PCR following Bmp2 and Runx2 mRNA expression. The cells’ inflammatory potential was assessed by qRT-PCR and ELISA following IL-1β, IL-6 and TNF-α expression and secretion. Activation of NFkB pathway was studied by analysing the phosphorylation of P65 via western blot. A siRNA approach was used to determine the signaling pathway involved in the effects of IS. RESULTS In pro-calcific conditions, IS increased hVICs osteogenic transition and calcification in a concentration-dependent manner, an effect that was blocked in hVICs transfected with siRNA targeting AhR expression. Data obtained by western blot showed that the exposure to [ISu] and [ISmax] promoted P65 phosphorylation in an AhR dependent manner. Blockade of the NFкB pathway with siRNA targeting P65 inhibited IS-induced expression of Bmp2 and RunX2 as well as hVICs mineralization, suggesting that hVICs inflammation plays a key role in the procalcific effects of IS. Confirming this hypothesis, the exposure to [ISu] and [ISmax] increased the expression of IL-1β, IL-6 and TNF-α transcripts, an effect abolished by silencing AhR and P65. Interestingly, hVICs did not secrete TNF-α and produced only a very low quantity of IL-1β in response to IS. By contrast, the exposure to [ISu] and [ISmax] induced an important release of IL-6 in hVICs culture medium, which was blocked when AhR and P65 were silenced. In this model, the use of an antibody neutralizing IL-6 completely abolished IS-induced hVICs osteogenic transition and calcification. CONCLUSION Our study demonstrates that IS displays direct pro-calcific effects on primary hVICs through AhR-dependent activation of the NFkB pathway and subsequent release of IL-6. We report that the autocrine action of IL-6 is responsible for IS-induced hVICs osteogenic transition and calcification. The observation that antibodies neutralizing IL-6 can block IS pro-calcific effects might be of particular interest knowing that interventional studies evaluating the cardiovascular impact of anti-IL-6 antibodies in patients with CKD are underway.

Examination of Genetic Control Elements in the Phototrophic Firmicute Heliomicrobium modesticaldum.

Heliomicrobium modesticaldum has been used as a model organism for the Heliobacteria, the only phototrophic family in the Firmicutes. It is a moderately thermophilic anoxygenic phototrophic bacterium that is capable of fermentative growth in the dark. The genetic manipulation of H. modesticaldum is still in its infancy. Methods to introduce genes through the use of exogenous plasmids and to delete genes from the chromosome through the use of the native CRISPR/Cas system have been developed in the last several years. To expand our genetic toolkit, it was necessary to control gene expression. In this study, we analyzed constitutive and inducible promoters developed for clostridia for their use in H. modesticaldum and further tested two reporters, adhB and lacZ, as indicators of promoter strength. Alcohol dehydrogenase (AdhB) was unsuitable as a reporter in this species due to high endogenous activity and/or low activity of the reporter, but a thermostable LacZ worked well as a reporter. A set of constitutive promoters previously reported to work in Clostridium thermocellum was found to be reliable for controlling the expression of the lacZ reporter gene in H. modesticaldum at a range of activities spanning an order of magnitude. An anhydrotetracycline-inducible promoter was created by inserting tetO operators into a strong constitutive promoter, but it was not fully repressible. The implementation of a xylose-inducible promoter resulted in complete repression of β-gal in the absence of xylose, and reliable expression tunable through the concentration of xylose added to the culture.

Pls1 Is a Peroxisomal Matrix Protein with a Role in Regulating Lysine Biosynthesis.

Peroxisomes host essential metabolic enzymes and are crucial for human health and survival. Although peroxisomes were first described over 60 years ago, their entire proteome has not yet been identified. As a basis for understanding the variety of peroxisomal functions, we used a high-throughput screen to discover peroxisomal proteins in yeast. To visualize low abundance proteins, we utilized a collection of strains containing a peroxisomal marker in which each protein is expressed from the constitutive and strong TEF2 promoter. Using this approach, we uncovered 18 proteins that were not observed in peroxisomes before and could show their metabolic and targeting factor dependence for peroxisomal localization. We focus on one newly identified and uncharacterized matrix protein, Ynl097c-b, and show that it localizes to peroxisomes upon lysine deprivation and that its localization to peroxisomes depends on the lysine biosynthesis enzyme, Lys1. We demonstrate that Ynl097c-b affects the abundance of Lys1 and the lysine biosynthesis pathway. We have therefore renamed this protein Pls1 for Peroxisomal Lys1 Stabilizing 1. Our work uncovers an additional layer of regulation on the central lysine biosynthesis pathway. More generally it highlights how the discovery of peroxisomal proteins can expand our understanding of cellular metabolism.

Heterologous Synthesis of Monacolin J by Reconstructing Its Biosynthetic Gene Cluster in Aspergillus niger.

Monacolin J (MJ), a key precursor of Lovastatin, could synthesize important statin drug simvastatin by hydrolyzing lovastatin and adding different side chains. In this study, to reduce the cumbersome hydrolysis of lovastatin to produce MJ in the native strain Aspergillus terreus, the MJ biosynthetic pathway genes (lovB, lovC, lovG, and lovA) were heterologously integrated into the genome of Aspergillus. niger CBS513.88 with strong promoters and suitable integration sites, via yeast 2μ homologous recombination to construct expression cassettes of long-length genes and CRISPR/Cas9 homology-directed recombination (CRISPR-HDR) to integrate MJ genes in the genome of A. niger. RT-PCR results proved that pathway synthesis-related genes could be heterologously expressed in A. niger. Finally, we constructed an engineered strain that could produce monacolin J, detected by LC-HR-ESIMS (MJ, 339.22 [M-H]+). The yield of MJ reached 92.90 mg/L after 7-day cultivation. By optimizing the cultivation conditions and adding precursor, the final titer of MJ was 142.61 mg/L on the fourth day of fed-batch cultivation, which was increased by 53.5% compared to the original growth conditions. Due to the wide application of A. niger in industrial fermentation for food and medicine, the following work will be dedicated to optimizing the metabolic network to improve the MJ production in the engineered strain.

Piperacillin/tazobactam-resistant, cephalosporin-susceptible Escherichia coli bloodstream infections are driven by multiple acquisition of resistance across diverse sequence types.

Resistance to piperacillin/tazobactam (TZP) in Escherichia coli has predominantly been associated with mechanisms that confer resistance to third-generation cephalosporins. Recent reports have identified E. coli strains with phenotypic resistance to piperacillin/tazobactam but susceptibility to third-generation cephalosporins (TZP-R/3GC-S). In this study we sought to determine the genetic diversity of this phenotype in E. coli (n=58) isolated between 2014–2017 at a single tertiary hospital in Liverpool, UK, as well as the associated resistance mechanisms. We compare our findings to a UK-wide collection of invasive E. coli isolates (n=1509) with publicly available phenotypic and genotypic data. These data sets included the TZP-R/3GC-S phenotype (n=68), and piperacillin/tazobactam and third-generation cephalosporin-susceptible (TZP-S/3GC-S, n=1271) phenotypes. The TZP-R/3GC-S phenotype was displayed in a broad range of sequence types, which was mirrored in the same phenotype from the UK-wide collection, and the overall diversity of invasive E. coli isolates. The TZP-R/3GC-S isolates contained a diverse range of plasmids, indicating multiple acquisition events of TZP resistance mechanisms rather than clonal expansion of a particular plasmid or sequence type. The putative resistance mechanisms were equally diverse, including hyperproduction of TEM-1, either via strong promoters or gene amplification, carriage of inhibitor-resistant β-lactamases, and an S133G bla CTX-M-15 mutation detected for the first time in clinical isolates. Several of these mechanisms were present at a lower abundance in the TZP-S/3GC-S isolates from the UK-wide collection, but without the associated phenotypic resistance to TZP. Eleven (19%) of the isolates had no putative mechanism identified from the genomic data. Our findings highlight the complexity of this cryptic phenotype and the need for continued phenotypic monitoring, as well as further investigation to improve detection and prediction of the TZP-R/3GC-S phenotype from genomic data.

Engineering and validation of a dual luciferase reporter system for quantitative and systematic assessment of regulatory sequences in Chinese hamster ovary cells

Ongoing research efforts to identify potent regulatory sequences that deliver robust and sustained transgene expression are critical for Chinese hamster ovary (CHO) cell line development technologies to meet the growing demand for recombinant proteins. Here we report the engineering and validation of a highly customizable single vector toolkit that comprises an all-in-one dual luciferase reporter system for quantitative and systematic interrogation of transcriptional regulatory sequences in transient and stable transfectants of CHO cells. To model the execution of the reporter system, we implemented a battery of known constitutive promoters including human CMV-mIE, SV40, HSV-TK, mouse PGK, human EF1α, EF1α short (EFS), human UBC, synthetic CAG, and Chinese hamster EF1α (CHEF1α). Of the nine promoters, CMV-mIE yielded the highest transcriptional activity in transient transfection settings, while CHEF1α was the strongest among a select subset of promoters in stable transfectants of CHO-DG44 pools. Remodeling the vector toolkit to build a dual fluorescent reporter system featured an alternative to bioluminescence based reporters. We infer that the findings of this study may serve as a basis to establish new vectors with weak or strong constitutive promoters. Furthermore, the modular all-in-one architecture of the reporter system proved to be a viable tool for discovering novel regulatory sequences that ensure high levels of transient and stable transgene expression in CHO and perhaps other mammalian cell lines.

Engineering cascade biocatalysis in whole cells for bottom-up synthesis of cello-oligosaccharides: flux control over three enzymatic steps enables soluble production

BackgroundSoluble cello-oligosaccharides (COS, β‐1,4‐D‐gluco‐oligosaccharides with degree of polymerization DP 2–6) have been receiving increased attention in different industrial sectors, from food and feed to cosmetics. Development of large-scale COS applications requires cost-effective technologies for their production. Cascade biocatalysis by the three enzymes sucrose-, cellobiose- and cellodextrin phosphorylase is promising because it enables bottom-up synthesis of COS from expedient substrates such as sucrose and glucose. A whole-cell-derived catalyst that incorporates the required enzyme activities from suitable co-expression would represent an important step towards making the cascade reaction fit for production. Multi-enzyme co-expression to reach distinct activity ratios is challenging in general, but it requires special emphasis for the synthesis of COS. Only a finely tuned balance between formation and elongation of the oligosaccharide precursor cellobiose results in the desired COS.ResultsHere, we show the integration of cellodextrin phosphorylase into a cellobiose-producing whole-cell catalyst. We arranged the co-expression cassettes such that their expression levels were upregulated. The most effective strategy involved a custom vector design that placed the coding sequences for cellobiose phosphorylase (CbP), cellodextrin phosphorylase (CdP) and sucrose phosphorylase (ScP) in a tricistron in the given order. The expression of the tricistron was controlled by the strong T7lacO promoter and strong ribosome binding sites (RBS) for each open reading frame. The resulting whole-cell catalyst achieved a recombinant protein yield of 46% of total intracellular protein in an optimal ScP:CbP:CdP activity ratio of 10:2.9:0.6, yielding an overall activity of 315 U/g dry cell mass. We demonstrated that bioconversion catalyzed by a semi-permeabilized whole-cell catalyst achieved an industrial relevant COS product titer of 125 g/L and a space–time yield of 20 g/L/h. With CbP as the cellobiose providing enzyme, flux into higher oligosaccharides (DP ≥ 6) was prevented and no insoluble products were formed after 6 h of conversion.ConclusionsA whole-cell catalyst for COS biosynthesis was developed. The coordinated co-expression of the three biosynthesis enzymes balanced the activities of the individual enzymes such that COS production was maximized. With the flux control set to minimize the share of insolubles in the product, the whole-cell synthesis shows a performance with respect to yield, productivity, product concentration and quality that is promising for industrial production.