Deficient Strain Research Articles

A Colletotrichum fructicola dual specificity phosphatase CfMsg5 is regulated by the CfAp1 transcription factor during oxidative stress and promotes virulence on Camellia oleifera

ABSTRACT Anthracnose, caused by Colletotrichum species, induces significant economic damages to crop plants annually, especially for Camellia oleifera. During infection, the counter-defence mechanisms of plant pathogens against ROS-mediated resistance, however, remain poorly understood. By employing Weighted Gene Co-expression Network Analysis (WGCNA), we identified ACTIVATOR PROTEIN-1 (AP-1), a bZIP transcription factor, as significant to infection. And deletion of CfAP1 inhibited aerial hyphae formation and growth under oxidative stress. Furthermore, RNA-seq analysis post H2O2 treatment revealed 33 significantly down-regulated genes in the AP-1 deficient strain, including A12032, a dual specificity phosphatase (DSP) homologous to MSG5 from Saccharomyces cerevisiae. This ΔCfmsg5 strain showed enhanced oxidative tolerance, reduced ROS scavenging, and negative regulation of the CWI MAPK cascade under oxygen stress, suggesting its involvement in oxidative signal transduction. Importantly, we provide evidence that CfMsg5 regulates growth, endoplasmic reticulum stress, and several unfolded protein response genes upregulated in ΔCfmsg5. Collectively, this study identified core components during C. fructicola infection and highlights a potential regulatory module involving CfAp1 and CfMsg5 in response to host ROS bursts. It provides new insights into fungal infection mechanisms and potential targets like CfAP1 and CfMSG5 for managing anthracnose diseases.

Convergent evolution links molybdenum insertase domains with organism-specific sequences

In all domains of life, the biosynthesis of the pterin-based Molybdenum cofactor (Moco) is crucial. Molybdenum (Mo) becomes biologically active by integrating into a unique pyranopterin scaffold, forming Moco. The final two steps of Moco biosynthesis are catalyzed by the two-domain enzyme Mo insertase, linked by gene fusion in higher organisms. Despite well-understood Moco biosynthesis, the evolutionary significance of Mo insertase fusion remains unclear. Here, we present findings from Neurospora crassa that shed light on the critical role of Mo insertase fusion in eukaryotes. Substituting the linkage region with sequences from other species resulted in Moco deficiency, and separate expression of domains, as seen in lower organisms, failed to rescue deficient strains. Stepwise truncation and structural modeling revealed a crucial 20-amino acid sequence within the linkage region essential for fungal growth. Our findings highlight the evolutionary importance of gene fusion and specific sequence composition in eukaryotic Mo insertases.

Butyrate as a growth factor of Clostridium acetobutylicum

The butyrate biosynthetic pathway not only contributes to electron management and energy generation in butyrate forming bacteria, but also confers evolutionary advantages to the host by inhibiting the growth of surrounding butyrate-sensitive microbes. While high butyrate levels induce toxic stress, effects of non-toxic levels on cell growth, health, metabolism, and sporulation remain unclear. Here, we show that butyrate stimulates cellular processes of Clostridium acetobutylicum, a model butyrate forming Firmicute. First, we deleted the 3-hydroxybutyryl-CoA dehydrogenase gene (hbd) from the C. acetobutylicum chromosome to eliminate the butyrate synthetic pathway and thus butyrate formation. A xylose inducible Cas9 cassette was chromosomally integrated and utilized for the one-step markerless gene deletions. Non-toxic butyrate levels significantly affected growth, health, and sporulation of C. acetobutylicum. After deleting spo0A, the gene encoding the master regulator of sporulation, Spo0A, and conducting butyrate addition experiments, we conclude that butyrate affects cellular metabolism through both Spo0A-dependent and independent mechanisms. We also deleted the hbd gene from the chromosome of the asporogenous C. acetobutylicum M5 strain lacking the pSOL1 plasmid to examine the potential involvement of pSOL1 genes on the observed butyrate effects. Addition of crotonate, the precursor of butyrate biosynthesis, to the hbd deficient M5 strain was used to probe the role of butyrate biosynthesis pathway in electron and metabolic fluxes. Finally, we found that butyrate addition can enhance the growth of the non-butyrate forming Clostridium saccharolyticum. Our data suggest that butyrate functions as a stimulator of cellular processes, like a growth factor, in C. acetobutylicum and potentially evolutionarily related Clostridium organisms.

Novel Role of Endothelial CD45 in Regulating Endothelial-to-Mesenchymal Transition in Atherosclerosis.

Protein-tyrosine-phosphatase CD45 is exclusively expressed in all nucleated cells of the hematopoietic system but is rarely expressed in endothelial cells. Interestingly, our recent study indicated that activation of the endogenous CD45 promoter in human endothelial colony forming cells (ECFCs) induced expression of multiple EndoMT marker genes. However, the detailed molecular mechanisms underlying CD45 that drive EndoMT and the therapeutic potential of manipulation of CD45 expression in atherosclerosis are entirely unknown. We generated a tamoxifen-inducible EC-specific CD45 deficient mouse strain (EC-iCD45KO) in an ApoE-deficient (ApoE-/-) background and fed with a Western diet (C57BL/6) for atherosclerosis and molecular analyses. We isolated and enriched mouse aortic endothelial cells with CD31 beads to perform single-cell RNA sequencing. Biomedical, cellular, and molecular approaches were utilized to investigate the role of endothelial CD45-specific deletion in the prevention of EndoMT in ApoE-/- model of atherosclerosis. Single-cell RNA sequencing revealed that loss of endothelial CD45 inhibits EndoMT marker expression and transforming growth factor-β signaling in atherosclerotic mice. which is associated with the reductions of lesions in the ApoE-/- mouse model. Mechanistically, the loss of endothelial cell CD45 results in increased KLF2 expression, which inhibits transforming growth factor-β signaling and EndoMT. Consistently, endothelial CD45 deficient mice showed reduced lesion development, plaque macrophages, and expression of cell adhesion molecules when compared to ApoE-/- controls. These findings demonstrate that the loss of endothelial CD45 protects against EndoMT-driven atherosclerosis, promoting KLF2 expression while inhibiting TGFβ signaling and EndoMT markers. Thus, targeting endothelial CD45 may be a novel therapeutic strategy for EndoMT and atherosclerosis.

Metformin reduced the alkaline resistance of Enterococcus faecalis against calcium hydroxide via Man-PTS EII: in vitro and in vivo studies.

The mannose phosphotransferase system (Man-PTS) plays crucial roles in the adaptive metabolic activity of Enterococcus faecalis (E. faecalis) in adverse environments. The aim of this study was to evaluate the role of Man-PTS in the alkaline resistance of E. faecalis against calcium hydroxide (CH) and the effect of metformin (Met) on the alkaline resistance of E. faecalis to CH. The regulatory role of Man-PTS EII in the alkaline resistance of E. faecalis was firstly investigated using a wild-type highly alkaline-resistant E. faecalis XS 003, standard ATCC 29212 and Man-PTS EIID gene deficient (△mptD) and overexpressing (+mptD) strains of E. faecalis. RNA sequencing of Met-treated E. faecalis was performed to further validate the effect of Met on Man-PTS. The effect of Met on CH resistance of E. faecalis was verified by evaluating the survival, membrane potential and permeability, intracellular pH and ATP, and the expression of Man-PTS EII and membrane transporter-related genes of E. faecalis. The effect of Met on the ability of CH to remove E. faecalis biofilm on the dentin surface was also tested. The in vivo therapeutic effect of Met plus CH (CHM) was further investigated in a rat apical periodontitis model induced by E. faecalis XS 003. Man-PTS EII significantly promoted the survival ability of E. faecalis in CH and enhanced its resistance to CH. The inhibition of Man-PTS EII by Met resulted in reduced alkaline resistance of E. faecalis in the presence of CH, while also enhancing the antimicrobial properties of CH against E. faecalis biofilm on dentin. Additionally, Met plus CH showed the synergistically promoted intra-canal E. faecalis infection control and healing of periapical lesion in rats. Met could significantly reduce the alkaline resistance of E. faecalis against CH through the modulation of Man-PTS EII, and improved the antibacterial effect of CH against E. faecalis infection both in vitro and in vivo. Met could significantly enhance the ability of CH to control E. faecalis infection through reducing the alkaline resistance of E. faecalis.

Effective enhancement of the ability of Monascus pilosus to produce lipid-lowering compound Monacolin K via perturbation of metabolic flux and histone acetylation modification

Monacolin K (MK), also known as lovastatin, is a polyketide compound with the ability to reduce plasma cholesterol levels and many other bio-activities. Red yeast rice (also named Hongqu) rich in MK derived from Monascus fermentation has attracted widespread attention due to its excellent performance in reducing blood lipids. However, industrial Monascus fermentation suffers from the limitations such as low yield of MK, long fermentation period, and susceptibility to contamination. In this study, we firstly blocked the competitive pathway of MK biosynthesis to create polyketide synthase gene pigA (the key gene responsible for the biosynthesis of Monascus azaphilone pigments) deficient strain A1. Then, based on the strategies to increase precursor supply for MK biosynthesis, acetyl-CoA carboxylase gene acc overexpression strains C1 and C2 were constructed with WT and A1 as the parent, respectively. Finally, histone deacetylase gene hos2 overexpression strain H1 was constructed by perturbation of histone acetylation modification. HPLC detection revealed all these four strains significantly increased their abilities to produce MK. After 14 days of solid-state fermentation, the MK yields of strains A1, C1, C2, and H1 reached 2.03 g/100 g, 1.81 g/100 g, 2.45 g/100 g and 2.52 g/100 g, which increased by 28.5 %, 14.7 %, 43.9 % and 36.1 % compared to WT, respectively. RT-qPCR results showed that overexpression of hos2 significantly increased the expression level of almost all genes responsible for MK biosynthesis after 5-day growth. Overall, the abilities of these strains to produce MK has been greatly improved, and MK production period has been shortened to 14 days from 20 days, providing new approaches for efficient production of Hongqu rich in MK.

Microbial intestinal dysbiosis drives long-term allergic susceptibility by sculpting an ILC2–B1 cell–innate IgE axis

BackgroundThe abundance and diversity of intestinal commensal bacteria influence systemic immunity with impact on disease susceptibility and severity. For example, loss of short chain fatty acid (SCFA)-fermenting bacteria in early life (humans and mice) is associated with enhanced type 2 immune responses in peripheral tissues including the lung. ObjectiveOur goal was to reveal the microbiome-dependent cellular and molecular mechanisms driving enhanced susceptibility to type 2 allergic lung disease. MethodsWe used low-dose vancomycin to selectively deplete SCFA-fermenting bacteria in wild type mice (Vanc-dys mice). We then examined the frequency and activation status of innate and adaptive immune cell lineages with and without SCFA supplementation. Finally, we used ILC2-deficient and signal transducer and activator of transcription 6 (STAT6)-deficient transgenic mouse strains to delineate the cellular and cytokine pathways leading to enhanced allergic disease susceptibility. ResultsVanc-dys mice exhibit a 2-fold increase in lung ILC2 primed to produce elevated levels of interleukin (IL)-2, -5 and -13. In addition, upon IL-33 treatment, Vanc-dys lung ILC2 display a novel ability to produce high levels of IL-4. These expanded and primed ILC2 drive B1 cell expansion and IL-4-dependent production of IgE that, in turn, leads to exacerbated allergic inflammation. Importantly, these enhanced lung inflammatory phenotypes in Vanc-dys mice were reversed by administration of dietary SCFA (specifically butyrate). ConclusionSCFA regulate an ILC2-B1cell-IgE axis. Early-life administration of vancomycin, an antibiotic known to deplete SCFA-fermenting gut bacteria, primes and amplifies this axis and leads to a lifelong enhanced susceptibility to type 2 allergic lung disease.

Construction of an expression platform for fungal secondary metabolite biosynthesis in Penicillium crustosum

Filamentous fungi are prolific producers of bioactive natural products and play a vital role in drug discovery. Yet, their potential cannot be fully exploited since many biosynthetic genes are silent or cryptic under laboratory culture conditions. Several strategies have been applied to activate these genes, with heterologous expression as one of the most promising approaches. However, successful expression and identification of new products are often hindered by host-dependent factors, such as low gene targeting efficiencies, a high metabolite background, or a lack of selection markers. To overcome these challenges, we have constructed a Penicillium crustosum expression host in a pyrG deficient strain by combining the split-marker strategy and CRISPR-Cas9 technology. Deletion of ligD and pcribo improved gene targeting efficiencies and enabled the use of an additional selection marker in P. crustosum. Furthermore, we reduced the secondary metabolite background by inactivation of two highly expressed gene clusters and abolished the formation of the reactive ortho-quinone methide. Finally, we replaced the P. crustosum pigment gene pcr4401 with the commonly used Aspergillus nidulans wA expression site for convenient use of constructs originally designed for A. nidulans in our P. crustosum host strain. As proof of concept, we successfully expressed a single polyketide synthase gene and an entire gene cluster at the P. crustosum wA locus. Resulting transformants were easily detected by their albino phenotype. With this study, we provide a highly efficient platform for heterologous expression of fungal genes.Key pointsConstruction of a highly efficient Penicillium crustosum heterologous expression hostReduction of secondary metabolite background by genetic dereplication strategyIntegration of wA site to provide an alternative host besides Aspergillus nidulansGraphical

The characteristics of autolysins associated with cell separation in Bacillus subtilis.

The peptidoglycan hydrolases responsible for the cell separation of Bacillus subtilis cells are collectively referred to as autolysins. However, the role of each autolysin in the cell separation of B. subtilis is not fully understood. In this study, we constructed a series of cell separation-associated autolysin deficient strains and strains overexpressing the transcription factors SlrR and SinR, and the morphological changes of these strains in liquid culture were observed. The results showed that the absence of D,L-endopeptidases CwlS and LytF only increased the cell chain length in the early exponential phase. The absence of D,L-endopeptidase LytE or N-acetylmuramyl-L-alanine amidase LytC can cause cells to form chains throughout the growth of B. subtilis, although the cell chain length was significantly shortened during the stationary phase. However, the absence of peptidoglycan N-acetylglucosaminidase LytD only caused minor defect in cell separation. Therefore, we concluded that LytE and LytC were the major autolysins that ensure the timely separation of B. subtilis daughter cells, whereas CwlS, LytF, and LytD were the minor autolysins. In addition, overexpression of the transcription factors SinR and SlrR in the cwlS lytF lytC lytE mutant enabled B. subtilis cells to form ultra-long chains in the vegetative phase, and its biomass level was basically the same as that of the wild type. This led to the conclusion that besides inhibiting the expression of lytC and lytF, the SinR-SlrR complex also has other potential mechanisms to inhibit cell separation.IMPORTANCEIn this study, the effects of CwlS, LytC, LytD, LytF, LytE, and SinR-SlrR complex on the cell separation of Bacillus subtilis at different growth phases were studied, and an ultra-long-chained B. subtilis strain was constructed. In microbial fermentation, due to its large cell size, this ultra-long-chained B. subtilis strain may be more likely to be precipitated or intercepted during the removal of bacterial process with centrifugation and membrane filtration as the main methods, which is crucial to improve the purity of the product.

Sialic acids from the Group B Streptococcus (GBS) capsule dampen mast cell activation through Siglec-9

Abstract GBS is a leading cause of bacterial infection in newborns, adult diabetics, pregnant women, and the elderly. A key virulence factor of GBS is its surface capsular polysaccharide, which displays terminal sialic acids that suppress host innate immune responses by engaging host inhibitory receptors like Sialic acid-binding immunoglobin-like lectin- 9 (Siglec-9). We reported that human mast cells (MC) express Siglec-9 and that engaging it with native ligands or antibodies results in reduced mast cell activation in vitro. We hypothesized that capsular sialic acids engage Siglec-9 to impair MC ability to initiate the host response. Mice expressing human Siglec -7 and -9 were infected with WT GBS or a sialic acid deficient strain, ΔcpsK, and the ΔcpsK infected mice showed reduced bacterial burden and less dissemination. Using bone marrow-cultured MC from a transgenic mouse expressing Siglec-9, we confirmed that GBS interacts with MC Siglec-9, which inhibited the release of the pro-inflammatory cytokine IL-6. Challenge of primary human MC with WT, ΔcpsK, or ΔneuA, another sialic acid deficient GBS strain, showed that MC interact more with ΔcpsK and ΔneuA GBS, and that MC kill sialic acid deficient GBS. MC challenged with ΔcpsK or ΔneuA GBS also produce more IL-8 and undergo cell death more than WT infected MC. Together, this data shows that sialylated GBS capsule engages MC Siglec-9 to dampen activation, which reduces the immune response and benefits the pathogen.

Abstract 3003: Ketogenic Diet Limits Experimental Abdominal Aortic Aneurysms

Background: Intermittent fasting and ketogenic diet may improve cardiovascular health. We studied the impact of ketogenic dietary intervention on experimental abdominal aortic aneurysms (AAAs). Methods: AAAs were induced in male mice via topical abluminal porcine pancreatic elastase (PPE) application at laparotomy in C57BL/6J, and 28 day administration of exogenous angiotensin (Ang) II in apolipoprotein E deficient strains, respectively. As feeding regimens, mice were fed standard or ketogenic diets 3 days prior to or 3 days following, AAA induction. Influence on AAA development was assessed by serial transabdominal ultrasonography and histopathologic analysis at sacrifice. Results: Ketogenic diet promotes cardiovascular health in part by increasing ketone body production. The Figure summarizes the influence of ketogenic diet on experimental AAAs in two distinct models. Following PPE application, subsequent aortic diameter enlargement was significantly reduced in mice receiving ketogenic vs. standard diets at 7 and 14 days regardless of specific feeding regimen. On histologic analysis, ketogenic diet feeding resulted in improved aortic medial elastin and smooth muscle cell retention, reduced mural macrophage, CD4 + and CD8 + T and B cell infiltration, and attenuated mural angiogenesis. In the Ang II infusion model, ketogenic diet limited Ang II-induced aortic diameter expansion, lowered AAA incidence and reduced AAA severity. Conclusion: Ketogenic dietary intervention suppressed experimental AAA progression in complementary murine modeling systems. Already popular clinically, this dietary modification may prove effective in limiting clinical AAA disease progression as well as other cardiovascular disease endpoints.

Deletion of Dictyostelium tpc2 gene forms multi-tipped structures, regulates autophagy and cell-type patterning.

Two pore channels (TPCs) are voltage-gated ion channel superfamily members that release Ca2+ from acidic intracellular stores and are ubiquitously present in both animals and plants. Starvation initiates multicellular development in Dictyostelium discoideum. Increased intracellular calcium levels bias Dictyostelium cells towards the stalk pathway and thus we decided to analyze the role of TPC2 in development, differentiation, and autophagy. We showed TPC2 protein localizes in lysosome-like acidic vesicles and the in situ data showed stalk cell biasness. Deletion of tpc2 showed defective and delayed development with formation of multi-tipped structures attached to a common base, while tpc2OE cells showed faster development with numerous small-sized aggregates and wiry fruiting bodies. The tpc2OE cells showed higher intracellular cAMP levels as compared to the tpc2- cells while pinocytosis was found to be higher in the tpc2- cells. Also, TPC2 regulates cell-substrate adhesion and cellular morphology. Under nutrient starvation, deletion of tpc2 reduced autophagic flux as compared to Ax2. During chimera formation, tpc2- cells showed a bias towards the prestalk/stalk region while tpc2OE cells showed a bias towards the prespore/spore region. tpc2 deficient strain exhibits aberrant cell-type patterning and loss of distinct boundary between the prestalk/prespore regions. TPC2 is required for effective development and differentiation in Dictyostelium and supports autophagic cell death and cell-type patterning. Decreased calcium due to deletion of tpc2 inhibit autophagic flux.

Abstract 1384 Manganese deficient strains of E. coli exhibit elevated rates of mutation

Abstract 1384 Manganese deficient strains of E. coli exhibit elevated rates of mutation

Deleting the C84L Gene from the Virulent African Swine Fever Virus SY18 Does Not Affect Its Replication in Porcine Primary Macrophages but Reduces Its Virulence in Swine.

(1) Background: African swine fever (ASF) is a highly contagious disease that causes high pig mortality. Due to the absence of vaccines, prevention and control are relatively challenging. The pathogenic African swine fever virus (ASFV) has a complex structure and encodes over 160 proteins, many of which still need to be studied and verified for their functions. In this study, we identified one of the unknown functional genes, C84L. (2) Methods: A gene deficient strain was obtained through homologous recombination and several rounds of purification, and its replication characteristics and virulence were studied through in vitro and in vivo experiments, respectively. (3) Results: Deleting this gene from the wild-type virulent strain SY18 did not affect its replication in porcine primary macrophages but reduced its virulence in pigs. In animal experiments, we injected pigs with a 102 TCID50, 105 TCID50 deletion virus, and a 102 TCID50 wild-type strain SY18 intramuscularly. The control group pigs reached the humane endpoint on the ninth day (0/5) and were euthanized. Two pigs in the 102 TCID50(2/5) deletion virus group survived on the twenty-first day, and one in the 105 TCID50(1/5) deletion virus group survived. On the twenty-first day, the surviving pigs were euthanized, which was the end of the experiment. The necropsies of the survival group and control groups' necropsies showed that the surviving pigs' liver, spleen, lungs, kidneys, and submaxillary lymph nodes did not show significant lesions associated with the ASFV. ASFV-specific antibodies were first detected on the seventh day after immunization; (4) Conclusions: This is the first study to complete the replication and virulence functional exploration of the C84L gene of SY18. In this study, C84L gene was preliminarily found not a necessary gene for replication, gene deletion strain SY18ΔC84L has similar growth characteristics to SY18 in porcine primary alveolar macrophages. The C84L gene affects the virulence of the SY18 strain.

Marker recycling in Lentinula edodes via 5-fluoroorotic acid counter-selection.

Shiitake (Lentinula edodes) contains various beneficial compounds and possesses several notable properties. However, there are few reports on its molecular breeding due to delay in development of its gene-modifying technology. Therefore, here, strain UV30 (pyrG -) was bred from the UV-irradiated protoplasts of strain M2. Strain UV30 was uracil-auxotrophic, and the phenylalanine residue in the active centre of orotidine-5-phosphate decarboxylase encoded by pyrG in the strain was substituted with a serine residue. Next, a recycling marker consisting of the upstream sequence of ku80, a repeat sequence (a portion of the downstream sequence of ku80), pyrG, and the downstream sequence of ku80 was introduced into the strain UV30. Consequently, the prototrophic strain ckp2-1, in which ku80 was replaced with the recycling marker, was obtained. After cultivation in complete medium, mycelia from the edges of ckp2-1 colonies were inoculated into a complete medium containing 5-Fluoroorotic acid (5-FOA). A 5-FOA-resistant strain KaM2, in which pyrG sequence was spliced from the recycling marker sequence via homologous recombination, was obtained. In this study, we developed the first marker recycling system for multigene targeting in L. edodes. Moreover, the resulting ∆ku80 strain may serve as a non-homologous end-joining deficient strain for further genetic manipulations.

Based on Transcriptome Sequencing of Cell Wall Deficient Strain, Research on Arabinosyltransferase Inhibition's Effect on the Synthesis of Cell Wall in Chlamydomonas reinhardtii.

To explore the key genes involved in cell wall synthesis and understand the molecular mechanism of cell wall assembly in the model alga-Chlamydomonas reinhardtii, transcriptome sequencing was used to discover the differentially expressed genes in the cell wall defective strain. In the glucose metabolism, lipid metabolism, and amino acid metabolism pathways, the gene expressions involved in the synthesis of cell wall functional components were analyzed. The results showed that in the cell wall defective strain, arabinosyltransferase gene (XEG113, RRA) related to synthesis of plant extensin and some cell wall structural protein genes (hyp, PHC19, PHC15, PHC4, PHC3) were up-regulated, 1,3-β-glucan synthase gene (Gls2) and endoglucanase gene (EG2) about synthesis and degradation of glycoskeleton were both mainly up-regulated. Then, ethambutol dihydrochloride, an arabinosyltransferase inhibitor, was found to affect the permeability of the cell wall of the normal strain, while the cell wall deficient strain was not affected. To further research the function of arabinosyltransferase, the RRA gene was inactivated by knockout in the normal cell wall algal strain. Through a combination of microscope observation and physiological index detection, it was found that the cell wall of the mutant strains showed reduced structure levels, suggesting that the structure and function of the cell wall glycoprotein were weakened. Therefore, arabinosyltransferase may affect the glycosylation modification of cell wall glycoprotein, further affecting the structure assembly of cell wall glycoprotein.

Production of d-glucaric acid with phosphoglucose isomerase-deficient Saccharomyces cerevisiae

d-Glucaric acid is a potential biobased platform chemical. Previously mainly Escherichia coli, but also the yeast Saccharomyces cerevisiae, and Pichia pastoris, have been engineered for conversion of d-glucose to d-glucaric acid via myo-inositol. One reason for low yields from the yeast strains is the strong flux towards glycolysis. Thus, to decrease the flux of d-glucose to biomass, and to increase d-glucaric acid yield, the four step d-glucaric acid pathway was introduced into a phosphoglucose isomerase deficient (Pgi1p-deficient) Saccharomyces cerevisiae strain. High d-glucose concentrations are toxic to the Pgi1p-deficient strains, so various feeding strategies and use of polymeric substrates were studied. Uniformly labelled 13C-glucose confirmed conversion of d-glucose to d-glucaric acid. In batch bioreactor cultures with pulsed d-fructose and ethanol provision 1.3 g d-glucaric acid L−1 was produced. The d-glucaric acid titer (0.71 g d-glucaric acid L−1) was lower in nitrogen limited conditions, but the yield, 0.23 g d-glucaric acid [g d-glucose consumed]−1, was among the highest that has so far been reported from yeast. Accumulation of myo-inositol indicated that myo-inositol oxygenase activity was limiting, and that there would be potential to even higher yield. The Pgi1p-deficiency in S. cerevisiae provides an approach that in combination with other reported modifications and bioprocess strategies would promote the development of high yield d-glucaric acid yeast strains.

Complexation and evolution of cis-prenyltransferase homologues in Cinnamomum kanehirae deduced from kinetic and functional characterizations.

Eukaryotic dehydrodolichyl diphosphate synthases (DHDDSs), cis-prenyltransferases (cis-PTs) synthesizing precursors of dolichols to mediate glycoprotein biosynthesis require partners, for eample Nus1 in yeast and NgBR in animals, which are cis-PTs homologues without activity but to boost the DHDDSs activity. Unlike animals, plants have multiple cis-PT homologues to pair or stand alone to produce various chain-length products with less known physiological roles. We chose Cinnamomum kanehirae, a tree that contains two DHDDS-like and three NgBR-like proteins from genome analysis, and found that one DHDDS-like protein acted as a homodimeric cis-PT to make a medium-chain C55 product, while the other formed heterodimeric complexes with either one of two NgBR homologues to produce longer-chain products. Both complexes were functional to complement the growth defect of the yeast rer2 deficient strain at a higher temperature. From the roles for the polyprenol and dolichol biosynthesis and sequence motifs, their homologues in various species were compared to reveal their possible evolutionary paths.

Abstract 16746: Novel Role of Endothelial CD45 in Regulating Endothelial-to-Mesenchymal Transition (EndMT) in Atherosclerosis

Introduction: Atherosclerosis is the leading cause of mortality in the United States and most other developed countries. Endothelial cell (EC) activation is a hallmark of the initiation and progression of atherosclerosis; identification of molecular markers of dysfunctional endothelium represents a biological challenge to developing new therapeutic targets. CD45 is a marker of hematopoietic cells, which is rarely expressed in ECs. However, recent studies indicated that CD45 is indispensable in driving the endothelial-to-mesenchymal transition (EndMT) following myocardial infarction. EndMT has also been implicated in the pathogenesis of atheroma progression. Thus, we aim to investigate whether endothelial CD45 contributes to the progression of atherosclerosis by facilitating the EndMT. Methods and results: In vitro , we used CRISPR/inactive Cas9 transcriptional activation system to express CD45 in human endothelial colony-forming cells (ECFCs). Using FACS analyses, we found that CD45+ECFCs upregulate transforming growth factors (TGFβ 1-3 ) that implicate EndMT but downregulate FGFR1 expression, which exacerbates EndMT. qRT-PCR and WB showed that CD45+ECFCs downregulate transcription factors KLFs. In vivo , we generated a tamoxifen-inducible EC-specific CD45 deficient mouse strain (EC-CD45iKO) in ApoE-deficient (ApoE-/-) background and fed with a Western diet (C57BL/6) for atherosclerosis and molecular analyses. We found that ApoE-/- / EC-iCD45KO mice reduced plaque macrophages, foam cells, expression of adhesion molecule, and lesion development compared to ApoE-/- controls. Interestingly, ApoE-/- / EC-iCD45KO mice showed the reduction of α-smooth muscle actin, which is typical of EndMT, in the aortic plaque. Single-cell RNA-seq analyses have been performed to characterize that endothelial CD45 regulates the expression of pro-inflammatory, EndMT, and endothelial cell markers genes on the aortic ECs. Conclusion: These findings demonstrate that endothelial CD45 potentiates atherosclerosis by inducing TGF signaling and mesenchymal phenotype, causing FGFR1 downregulation and attenuating KLFs expression to drive EndMT, highlighting endothelial CD45 may be a new drug target for the treatment of atherosclerosis.

Mitochondrial tryparedoxin peroxidase in the response of Trypanosoma cruzi to stress: characterization of deficient strains obtained by CRISPR/Cas9

Mitochondrial tryparedoxin peroxidase in the response of Trypanosoma cruzi to stress: characterization of deficient strains obtained by CRISPR/Cas9