Microbial chassis design and engineering for production of gamma-aminobutyric acid.

Gamma-aminobutyric acid (GABA) is a non-protein amino acid which is widely applied in agriculture and pharmaceutical additive industries. GABA is synthesized from glutamate through irreversible α-decarboxylation by glutamate decarboxylase. Recently, microbial synthesis has become an inevitable trend to produce GABA due to its sustainable characteristics. Therefore, reasonable microbial platform design and metabolic engineering strategies for improving production of GABA are arousing a considerable attraction. The strategies concentrate on microbial platform optimization, fermentation process optimization, rational metabolic engineering as key metabolic pathway modification, promoter optimization, site-directed mutagenesis, modular transporter engineering, and dynamic switch systems application. In this review, the microbial producers for GABA were summarized, including lactic acid bacteria, Corynebacterium glutamicum, and Escherichia coli, as well as the efficient strategies for optimizing them to improve the production of GABA.

Enhancing Protein Function Prediction Performance by Utilizing AlphaFold-Predicted Protein Structures.

The structure of a protein is of great importance in determining its functionality, and this characteristic can be leveraged to train data-driven prediction models. However, the limited number of available protein structures severely limits the performance of these models. AlphaFold2 and its open-source data set of predicted protein structures have provided a promising solution to this problem, and these predicted structures are expected to benefit the model performance by increasing the number of training samples. In this work, we constructed a new data set that acted as a benchmark and implemented a state-of-the-art structure-based approach for determining whether the performance of the function prediction model can be improved by putting additional AlphaFold-predicted structures into the training set and further compared the performance differences between two models separately trained with real structures only and AlphaFold-predicted structures only. Experimental results indicated that structure-based protein function prediction models could benefit from virtual training data consisting of AlphaFold-predicted structures. First, model performances were improved in all three categories of Gene Ontology terms (GO terms) after adding predicted structures as training samples. Second, the model trained only on AlphaFold-predicted virtual samples achieved comparable performances to the model based on experimentally solved real structures, suggesting that predicted structures were almost equally effective in predicting protein functionality.

Core Oligosaccharide Portion of Lipopolysaccharide Plays Important Roles in Multiple Antibiotic Resistance in Escherichia coli.

Gram-negative bacteria are intrinsically resistant to antibiotics due to the presence of the cell envelope, but the mechanisms of this resistance are still not fully understood. In this study, a series of mutants that lack one or more major components associated with the cell envelope were constructed from Escherichia coli K-12 W3110. WJW02 can only synthesize Kdo2-lipid A, which lacks the core oligosaccharide portion of lipopolysaccharide (LPS). WJW04, WJW07, and WJW08 were constructed from WJW02 by deleting the gene clusters relevant to the biosynthesis of exopolysaccharide, flagella, and fimbriae, respectively. WJW09, WJW010, and WJW011 cells cannot synthesize exopolysaccharide (EPS), flagella, and fimbria, respectively. Compared to the wild type (W3110), mutants WJW02, WJW04, WJW07, and WJW08 cells showed decreased resistance to more than 10 different antibacterial drugs, but the mutants WJW09, WJW010, and WJW011 did not. This indicates that the core oligosaccharide portion of lipopolysaccharide plays an important role in multiple antibiotic resistance in E. coli and that the first heptose in the core oligosaccharide portion is critical. Furthermore, the removal of the core oligosaccharide of LPS leads to influences on cell wall morphology, cell phenotypes, porins, efflux systems, and response behaviors to antibiotic stimulation. The results demonstrate the important role of lipopolysaccharide in the antibiotic resistance of Gram-negative bacteria.

Engineering the Outer Membrane Could Facilitate Better Bacterial Performance and Effectively Enhance Poly-3-Hydroxybutyrate Accumulation.

Poly-3-hydroxybutyrate (PHB) is an environmentally friendly polymer and can be produced in Escherichia coli cells after overexpression of the heterologous gene cluster phaCAB. The biosynthesis of the outer membrane (OM) consumes many nutrients and influences cell morphology. Here, we engineered the OM by disrupting all gene clusters relevant to the polysaccharide portion of lipopolysaccharide (LPS), colanic acid (CA), flagella, and/or fimbria in E. coli W3110. All these disruptions benefited PHB production. Especially, disrupting all these OM components increased the PHB content to 83.0 wt% (PHB content percentage of dry cell weight), while the wild-type control produced only 1.5 wt% PHB. The increase was mainly due to the LPS truncation to Kdo2 (3-deoxy-d-manno-octulosonic acid)-lipid A, which resulted in 82.0 wt% PHB with a 25-fold larger cell volume, and disrupting CA resulted in 57.8 wt% PHB. In addition, disrupting LPS facilitated advantageous fermentation features, including 69.1% less acetate, a 550% higher percentage of autoaggregated cells among the total culture cells, 69.1% less biofilm, and a higher broken cell ratio. Further detailed mechanism investigations showed that disrupting LPS caused global changes in envelope and cellular metabolism: (i) a sharp decrease in flagella, fimbria, and secretions; (ii) more elastic cells; (iii) much greater carbon flux toward acetyl coenzyme A (acetyl-CoA) and supply of cofactors, including NADP, NAD, and ATP; and (iv) a decrease in by-product acids but increase in γ-aminobutyric acid by activating σE factor. Disrupting CA, flagella, and fimbria also improved the levels of acetyl-CoA and cofactors. The results indicate that engineering the OM is an effective strategy to enhance PHB production and highlight the applicability of OM engineering to increase microbial cell factory performance. IMPORTANCE Understanding the detailed influence of the OM on the cell envelope and cellular metabolism is important for optimizing the E. coli cell factory and many other microorganisms. This study revealed the applicability of remodeling the OM to enhance PHB accumulation as representative inclusion bodies. The results generated in this study give essential information for producing other inclusion bodies or chemicals which need more acetyl-CoA and cofactors but less by-product acids. This study is promising to provide new ideas for the improvement of microbial cell factories.

Insights into the structure of Escherichia coli outer membrane as the target for engineering microbial cell factories.

Escherichia coli is generally used as model bacteria to define microbial cell factories for many products and to investigate regulation mechanisms. E. coli exhibits phospholipids, lipopolysaccharides, colanic acid, flagella and type I fimbriae on the outer membrane which is a self-protective barrier and closely related to cellular morphology, growth, phenotypes and stress adaptation. However, these outer membrane associated molecules could also lead to potential contamination and insecurity for fermentation products and consume lots of nutrients and energy sources. Therefore, understanding critical insights of these membrane associated molecules is necessary for building better microbial producers. Here the biosynthesis, function, influences, and current membrane engineering applications of these outer membrane associated molecules were reviewed from the perspective of synthetic biology, and the potential and effective engineering strategies on the outer membrane to improve fermentation features for microbial cell factories were suggested.

Impact of triglyceride-glucose index on long-term cardiovascular outcomes in patients with myocardial infarction with nonobstructive coronary arteries.

BACKGROUND AND AIMS: Triglyceride-glucose (TyG) index has been reported as a novel surrogate marker of insulin resistance and a risk factor in patients with coronary artery disease. We aimed to investigate the prognostic value of TyG index in a distinct entity with myocardial infarction with nonobstructive coronary arteries (MINOCA). METHODS AND RESULTS: A total of 1179 MINOCA patients were recruited and divided according to tertile levels of TyG index. The primary endpoint was a composite of major adverse cardiovascular events (MACE), including all-cause death, reinfarction, stroke, revascularization and hospitalization for unstable angina or heart failure. Kaplan-Meier, Cox regression and receiver-operating characteristic analyses were performed. Patients with higher tertiles of TyG index had a significantly higher incidence of MACE (9.6%, 14.9%, 18.0%; p = 0.003) over the median follow-up of 41.7 months. After multivariate adjustment, elevated TyG index was significantly associated with an increased risk of MACE (HR 1.33, 95% CI: 1.04-1.69, p = 0.020). The adjusted risk of MACE also increased with rising tertiles of TyG index (tertile 1 as reference; tertile 2: HR 1.64, 95% CI: 1.06-2.53, p = 0.025; tertile 3: HR 1.85, 95% CI: 1.17-2.93, p = 0.008). The TyG index remained a robust risk factor in overall and subgroups of MINOCA patients (all p < 0.05). Moreover, the TyG index yielded a moderate predictive value of MACE (area under the curve 0.66, 95% CI:0.61-0.71, p < 0.001). CONCLUSION: Elevated TyG index was independently associated with a poor prognosis after MINOCA. Routine assessment of TyG index may improve risk stratification and facilitate decision making in MINOCA patients.

Metabolic engineering of Corynebacterium glutamicum WM001 to improve l-isoleucine production.

In this study, l-isoleucine production in Corynebacterium glutamicum WM001 was improved by deleting three genes in the genome, replacing the native promoter of ilvA in the genome, and overexpression of five genes in an alr-based auxotrophic complementation expression system. The three genes deleted in the genome are alaT, brnQ, and alr. Deletion of alaT improved l-isoleucine production by increasing the supply of pyruvate, whereas deletion of brnQ improved l-isoleucine production by blocking the uptake of extracellular l-isoleucine. Exchange of the native promoter of ilvA with promoter tac or tacM could contribute to l-isoleucine production by increasing 2-ketobutyric acid; tac is better than tacM for improving l-isoleucine yield. Different combinations of the genes ilvBN, ppnK, lrp, and brnFE were overexpressed in an alr-based auxotrophic complementation expression system to further improve l-isoleucine production, and the best yield after 72-H flask fermentation was obtained from the strain WM005/pYCW-1-ilvBN2-ppnK1. Without addition of any antibiotics, WM005/pYCW-1-ilvBN2-ppnK1 could produce 32.1 g/L l-isoleucine after 72-H fed-batch fermentation, which is 34.3% increase compared with the original strain WM001.

Identification of Key Genes and Underlying Mechanisms in Acute Kawasaki Disease Based on Bioinformatics Analysis.

BACKGROUND Kawasaki disease (KD) is a systemic vasculitis that predominantly occurs in children, but the pathogenesis of KD remains unclear. Here, we explored key genes and underlying mechanisms potentially involved in KD using bioinformatic analyses. MATERIAL AND METHODS The shared differentially expressed genes (DEGs) in KD compared to control samples were identified using the microarray data from the Gene Expression Omnibus Series (GSE) 18606, GSE68004, and GSE73461. Analyses of the functional annotation, protein-protein interaction (PPI) network, microRNA-target DEGs regulatory network, and immune cell infiltration were performed. The expression of hub genes before and after intravenous immunoglobulin (IVIG) treatment in KD was further verified using GSE16797. RESULTS A total of 195 shared DEGs (164 upregulated and 31 downregulated genes) were identified between KD and healthy controls. These shared DEGs were mainly enriched in immune and inflammatory responses. Ten upregulated hub genes (ITGAX, SPI1, LILRB2, MMP9, S100A12, C3AR1, RETN, MAPK14, TLR5, MYD88) and the most significant module were identified in the PPI network. There were 309 regulatory relationships detected within 70 predicted microRNAs and 193 target DEGs. The immune cell infiltration analysis showed that monocytes, neutrophils, activated mast cells, and activated natural killer cells had relatively high proportions and were significantly more infiltrated in KD samples. Six hub genes of ITGAX, LILRB2, C3AR1, MAPK14, TLR5, and MYD88 were markedly downregulated after IVIG treatment for KD. CONCLUSIONS Our study identified the candidate genes and associated molecules that may be related to the KD process, and provided new insights into potential mechanisms and therapeutic targets for KD.

Human Papillomavirus E6/E7 and Long Noncoding RNA TMPOP2 Mutually Upregulated Gene Expression in Cervical Cancer Cells.

TMPOP2 was previously suggested to be an oncogenic long noncoding RNA which is excessively expressed in cervical cancer cells and inhibits E-cadherin gene expression by recruiting transcription repressor EZH2 to the gene promoter. So far, the function and regulation of TMPOP2 in cervical cancer remain largely unknown. Herein, we found that TMPOP2 expression was correlated with human papillomavirus 16/18 (HPV16/18) E6 and E7 in cervical cancer cell lines CaSki and HeLa. Tumor suppressor p53, which is targeted for degradation by HPV16/18, was demonstrated to associate with two p53 response elements in the TMPOP2 promoter to repress the transcription of the TMPOP2 gene. Reciprocally, ectopic expression of TMPOP2 was demonstrated to sequester tumor repressor microRNAs (miRNAs) miR-375 and miR-139 which target HPV16/18 E6/E7 mRNA and resulted in an upregulation of HPV16/18 E6/E7 genes. Thereby, HPV16/18 E6/E7 and the long noncoding RNA (lncRNA) TMPOP2 form a positive feedback loop to mutually derepress gene expression in cervical cancer cells. Moreover, results of RNA sequencing and cell cycle analysis showed that knockdown of TMPOP2 impaired the expression of cell cycle genes, induced cell cycle arrest, and inhibited HeLa cell proliferation. Together, our results indicate that TMPOP2 and HPV16/18 E6/E7 mutually strengthen their expression in cervical cancer cells to enhance tumorigenic activities.IMPORTANCE Human papillomaviruses 16 and 18 (HPV16/18) are the main causative agents of cervical cancer. Viral proteins HPV16/18 E6 and E7 are constitutively expressed in cancer cells to maintain oncogenic phenotypes. Accumulating evidences suggest that HPVs are correlated with the deregulation of long noncoding RNAs (lncRNAs) in cervical cancer, although the mechanism was unexplored in most cases. TMPOP2 is a newly identified lncRNA excessively expressed in cervical cancer. However, the mechanism for the upregulation of TMPOP2 in cervical cancer cells remains largely unknown and its relationship with HPVs is still elusive. The significance of our research is in revealing the mutual upregulation of HPV16/18 E6/E7 and TMPOP2 with the molecular mechanisms explored. This study will expand our understandings of the oncogenic activities of human papillomaviruses and lncRNAs.

Rebalancing microbial carbon distribution for L-threonine maximization using a thermal switch system.

In metabolic engineering, unbalanced microbial carbon distribution has long blocked the further improvement in yield and productivity of high-volume natural metabolites. Current studies mostly focus on regulating desired biosynthetic pathways, whereas few strategies are available to maximize L-threonine efficiently. Here, we present a strategy to guarantee the supply of reduced cofactors and actualize L-threonine maximization by regulating cellular carbon distribution in central metabolic pathways. A thermal switch system was designed and applied to divide the whole fermentation process into two stages: growth and production. This system could rebalance carbon substrates between pyruvate and oxaloacetate by controlling the heterogenous expression of pyruvate carboxylase and oxaloacetate decarboxylation that responds to temperature. The system was tested in an L-threonine producer Escherichia coli TWF001, and the resulting strain TWF106/pFT24rp overproduced L-threonine from glucose with 111.78% molar yield. The thermal switch system was then employed to switch off the L-alanine synthesis pathway, resulting in the highest L-threonine yield of 124.03%, which exceeds the best reported yield (87.88%) and the maximum available theoretical value of L-threonine production (122.47%). This inducer-free genetic circuit design can be also developed for other biosynthetic pathways to increase product conversion rates and shorten production cycles.

Expression regulation of multiple key genes to improve L-threonine in Escherichia coli.

BACKGROUND: Escherichia coli is an important strain for L-threonine production. Genetic switch is a ubiquitous regulatory tool for gene expression in prokaryotic cells. To sense and regulate intracellular or extracellular chemicals, bacteria evolve a variety of transcription factors. The key enzymes required for L-threonine biosynthesis in E. coli are encoded by the thr operon. The thr operon could coordinate expression of these genes when L-threonine is in short supply in the cell. RESULTS: The thrL leader regulatory elements were applied to regulate the expression of genes iclR, arcA, cpxR, gadE, fadR and pykF, while the threonine-activating promoters PcysH, PcysJ and PcysD were applied to regulate the expression of gene aspC, resulting in the increase of L-threonine production in an L-threonine producing E. coli strain TWF001. Firstly, different parts of the regulator thrL were inserted in the iclR regulator region in TWF001, and the best resulting strain TWF063 produced 16.34 g L-threonine from 40 g glucose after 30 h cultivation. Secondly, the gene aspC following different threonine-activating promoters was inserted into the chromosome of TWF063, and the best resulting strain TWF066 produced 17.56 g L-threonine from 40 g glucose after 30 h cultivation. Thirdly, the effect of expression regulation of arcA, cpxR, gadE, pykF and fadR was individually investigated on L-threonine production in TWF001. Finally, using TWF066 as the starting strain, the expression of genes arcA, cpxR, gadE, pykF and fadR was regulated individually or in combination to obtain the best strain for L-threonine production. The resulting strain TWF083, in which the expression of seven genes (iclR, aspC, arcA, cpxR, gadE, pykF, fadR and aspC) was regulated, produced 18.76 g L-threonine from 30 g glucose, 26.50 g L-threonine from 40 g glucose, or 26.93 g L-threonine from 50 g glucose after 30 h cultivation. In 48 h fed-batch fermentation, TWF083 could produce 116.62 g/L L-threonine with a yield of 0.486 g/g glucose and productivity of 2.43 g/L/h. CONCLUSION: The genetic engineering through the expression regulation of key genes is a better strategy than simple deletion of these genes to improve L-threonine production in E. coli. This strategy has little effect on the intracellular metabolism in the early stage of the growth but could increase L-threonine biosynthesis in the late stage.

Transcriptomic analysis of an l-threonine-producing Escherichia coli TWF001.

Wild-type Escherichia coli usually does not accumulate l-threonine, but E. coli strain TWF001 could produce 30.35 g/L l-threonine after 23-H fed-batch fermentation. To understand the mechanism for the high yield of l-threonine production in TWF001, transcriptomic analyses of the TWF001 cell samples collected at the logarithmic and stationary phases were performed, using the wild-type E. coli strain W3110 as the control. Compared with W3110, 1739 and 2361 genes were differentially transcribed in the logarithmic and stationary phases, respectively. Most genes related to the biosynthesis of l-threonine were significantly upregulated. Some key genes related to the NAD(P)H regeneration were upregulated. Many genes relevant to glycolysis and TCA cycle were downregulated. The key genes involved in the l-threonine degradation were downregulated. The gene rhtA encoding the l-threonine exporter was upregulated, whereas the genes sstT and tdcC encoding the l-threonine importer were downregulated. The upregulated genes in the glutamate pathway might form an amino-providing loop, which is beneficial for the high yield of l-threonine production. Many genes encoding the 30S and 50S subunits of ribosomes were also upregulated. The findings are useful for gene engineering to increase l-threonine production in E. coli.

DNMT3A inhibits E2F1-induced arterial marker expression and impairs angiogenesis in human umbilical artery endothelial cells.

Arterial marker genes EphrinB2 and HEY2 are essential for cardiovascular development and postnatal neovascularization. Our previous study confirmed that E2F1 could activate the transcription of EphrinB2 and HEY2 in human mesenchymal stem cells; however, the detailed mechanism has not been resolved yet. In this study, we focused on the interaction between E2F1 and DNMT3A, a de novo DNA methyltransferase, on regulating the expression of EphrinB2 and HEY2, and explored the potential mechanisms. Gain- and loss-of-function experiments implicated the positive effect of E2F1 on the expression of EphrinB2 and HEY2 and tube formation in human umbilical artery endothelial cells. Accumulation of DNMT3A decreased the levels of EphrinB2 and HEY2, and impaired tube formation induced by E2F1, while inhibiting DNMT3A by RNA interference augmented their expression and angiogenesis in E2F1-trasfected cells. We then asked whether the low expressions of EphrinB2 and HEY2 induced by DNMT3A are related to the methylation status of their promoters. Surprisingly, the methylation status of the CpG islands in the promoter region was not significantly affected by overexpression of exogenous DNMT3A. Furthermore, the interaction between E2F1 and DNMT3A was confirmed by co-immunoprecipitation. DNMT3A could inhibit the transcription of EphrinB2 and HEY2 promoters by affecting the binding of E2F1 to its recognition sequences as revealed by luciferase reporter assay and chromatin immunoprecipitation. These results identified a novel mechanism underlying the cooperation of DNMT3A with E2F1 on regulating target gene expression, and revealed their roles in the angiogenic process.

Cysteine synthase A overexpression in Corynebacterium glutamicum enhances l-isoleucine production.

Cysteine synthase A (CysK) catalyzes the last reaction of l-cysteine synthesis in bacteria, but its moonlighting functions have been revealed recently. In this study, CysK was overexpressed in Corynebacterium glutamicum IWJ001, an l-isoleucine producer. Compared with the control IWJ001/pDXW-8, IWJ001/pDXW-8-cysK cells grew fast during log phase, and produced 26.5% more l-isoleucine in flask fermentation and 23.5% more l-isoleucine in fed-batch fermentation. The key genes aspC, lysC, hom, thrB, ilvA, and ilvBN involved in l-isoleucine biosynthesis were all upregulated in IWJ001/pDXW-8-cysK, compared with IWJ001/pDXW-8. In addition, IWJ001/pDXW-8-cysK cells were longer and thicker than IWJ001/pDXW-8 cells. Compared with IWJ001/pDXW-8, the membrane permeability increased 15.8% and biofilm formation ability decreased 71.3% for IWJ001/pDXW-8-cysK cells. The results demonstrate that CysK overexpression in C. glutamicum is a good approach to enhance l-isoleucine production.

Increasing L-threonine production in Escherichia coli by overexpressing the gene cluster phaCAB.

L-Threonine is an important branched-chain amino acid and could be applied in feed, drugs, and food. In this study, L-threonine production in an L-threonine-producing Escherichia coli strain TWF001 was significantly increased by overexpressing the gene cluster phaCAB from Ralstonia eutropha. TWF001/pFW01-phaCAB could produce 96.4-g/L L-threonine in 3-L fermenter and 133.5-g/L L-threonine in 10-L fermenter, respectively. In addition, TWF001/pFW01-phaCAB produced 216% more acetyl-CoA, 43% more malate, and much less acetate than the vector control TWF001/pFW01, and meanwhile, TWF001/pFW01-phaCAB produced poly-3-hydroxybutyrate, while TWF001/pFW01 did not. Transcription analysis showed that the key genes in the L-threonine biosynthetic pathway were up-regulated, the genes relevant to the acetate formation were down-regulated, and the gene acs encoding the enzyme which converts acetate to acetyl-CoA was up-regulated. The results suggested that overexpression of the gene cluster phaCAB in E. coli benefits the enhancement of L-threonine production.

A Perfused Microfluidic System to Study the Differentiation of Neural Stem Cells in vitro.

INTRODUCTION: Due to the ability to mimic in vivo cellular microenvironments, the development of multicell culture systems has received increasing interest for use as research models and serving as platforms for drug evaluation. METHODS: In this study, we developed a perfused microfluidic system to resemble the in vivo intercellular environment and applied it to study the differentiation from neural stem cells into neurons. RESULTS: As determined by immunofluorescence chemistry and quantitative real-time PCR, the neural stem cells grown in this microfluidic system showed an elevated differentiation rate toward the formation of neurons as determined by the increased level of ßIII-tubulin production, which is 4 times higher than that of culturing neural stem cells only. CONCLUSION: These results revealed that some factors secreted into the intercellular microenvironment by adult neuron cells can stimulate the differentiation of neural stem cells, pointing to the importance of developing multicellular culture systems such as the perfused microfluidic system we report here to better resemble the in vivo situation.

Poly(3-hydroxybutyrate-co-3-hydroxyvalerate) co-produced with L-isoleucine in Corynebacterium glutamicum WM001.

BACKGROUND: Co-production of polyhydroxyalkanoate (PHA) and amino acids makes bacteria effective microbial cell factories by secreting amino acids outside while accumulating PHA granules inside. Poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) is one of the PHAs with biocompatibility and fine mechanical properties, but its production is limited by the low level of intracellular propionyl-CoA. RESULTS: L-Isoleucine producing Corynebacterium glutamicum strain WM001 were analyzed by genome and transcriptome sequencing. The results showed that the most over-expressed genes in WM001 are relevant not only to L-isoleucine production but also to propionyl-CoA accumulation. Compared to the wild-type C. glutamicum ATCC13869, the transcriptional levels of the genes prpC2, prpD2, and prpB2, which are key genes relevant to propionyl-CoA accumulation, increased 26.7, 25.8, and 28.4-folds in WM001, respectively; and the intracellular level of propionyl-CoA increased 16.9-fold in WM001. When the gene cluster phaCAB for PHA biosynthesis was introduced into WM001, the recombinant strain WM001/pDXW-8-phaCAB produced 15.0 g/L PHBV with high percentage of 3-hydroxyvalerate as well as 29.8 g/L L-isoleucine after fed-batch fermentation. The maximum 3-hydroxyvalerate fraction in PHBV produced by WM001/pDXW-8-phaCAB using glucose as the sole carbon source could reach 72.5%, which is the highest reported so far. CONCLUSIONS: Genome and transcriptome analysis showed that C. glutamicum WM001 has potential to accumulate L-isoleucine and propionyl-CoA pool. This was experimentally confirmed by introducing the phaCAB gene cluster into WM001. The recombinant strain WM001/pDXW-8-phaCAB produced high levels of PHBV with high 3-hydroxyvalerate fraction as well as L-isoleucine. Because of its high level of intracellular propionyl-CoA pool, WM001 might be used for producing other propionyl-CoA derivatives.

Deletion of 76 genes relevant to flagella and pili formation to facilitate polyhydroxyalkanoate production in Pseudomonas putida.

Pseudomonas putida KT2442, a natural producer of polyhydroxyalkanoate, spends a lot of energy and carbon sources to form flagella and pili; therefore, deleting the genes involved in the biosynthesis and assembly of flagella and pili might improve PHA productivity. In this study, two novel deletion systems were constructed in order to efficiently remove the 76 genes involved in the biosynthesis and assembly of flagella and pili in P. putida KT2442. Both systems combine suicide-plasmid-based homologous recombination and mutant lox site-specific recombination and involve three plasmids. The first includes pK18mobsacB, pWJW101, and pWJW102; and the second includes pZJD29c, pDTW202, and pWJW103. These newly constructed systems were successfully used to remove different gene clusters in P. putida KT2442 and showed a high deletion efficiency (above 90%) whether for the second-round or the third-round recombination. Both systems could efficiently delete the gene PP4378 encoding flagellin in putida KT2442, resulting in the mutant strain WJPP01. The second system was used to remove the pili-forming gene cluster PP2357-PP2363 in putida KT2442, resulting in the mutant strain WJPP02, and also used to remove the flagella-forming gene cluster PP4329-PP4397 in WJPP02, resulting in the mutant strain WJPP03. Compared with the wild-type KT2442, the 1.2% genome reduction mutant WJPP03 grew faster, lacked flagella and motility, showed sharply decreased biofilm and 3',5'-cyclic diguanylic acid (c-di-GMP), but accumulated more polyhydroxyalkanoate. The biomass, polyhydroxyalkanoate yield, and content of WJPP03 increased 19.1, 73.4, and 45.6%, respectively, with sodium hexanoate supplementation, and also increased 11.4, 53.6, and 37.9%, respectively, with lauric acid supplementation.

Knockdown of DNMT1 and DNMT3a Promotes the Angiogenesis of Human Mesenchymal Stem Cells Leading to Arterial Specific Differentiation.

Human mesenchymal stem cells (hMSCs) possess the potential to differentiate into endothelial cells (EC). DNA methylation plays an important role in cell differentiation during development. However, the role of the DNA methyltransferases Dnmt1 and Dnmt3a in specific arterial differentiation of hMSCs is not clear. Here, we show that the CpG islands in the promoter regions of the EC specification and arterial marker genes were highly methylated in hMSCs based on bisulfite genomic sequencing. Treatment with the DNMT inhibitor 5-aza-dc induced the reactivation of EC specification and arterial marker genes by promoting demethylation of these genes as well as stimulating tube-like structure formation. The hMSCs with stable knockdown of Dnmt1/Dnmt3a were highly angiogenic and expressed several arterial specific transcription factors and marker genes. A Matrigel plug assay confirmed that Dnmt1/Dnmt3a stable knockdown hMSCs enhanced blood vessel formation compared with WT MSCs. We also identified that the transcription factor E2F1 could upregulate the transcription of arterial marker genes by binding to the promoters of arterial genes, suggesting its critical role for arterial specification. Moreover, miRNA gain/loss-of-function analyses revealed that miR152 and miR30a were involved in endothelial differentiation of hMSCs by targeting Dnmt1 and Dnmt3a, respectively. Taken together, these data suggest that Dnmt1 and Dnmt3a are critical regulators for epigenetic silencing of EC marker genes and that E2F1 plays an important role in promoting arterial cell determination. Stem Cells 2016;34:1273-1283.

Heterologous expression in Pichia pastoris and characterization of a novel GH11 xylanase from saline-alkali soil with excellent tolerance to high pH, high salt concentrations and ethanol.

A GH11 xylanase gene (xyn11-1) cloned from saline-alkali soil was successfully expressed in Pichia pastoris GS115. The purified recombinant Xyn11-1 showed its maximal activity at pH 6.0, and retained more than 60.4% of activity at pH 10.0, with good pH stability. Its optimal temperature was 50 °C and it was stable after incubation for 1 h at 30 °C. Furthermore, Xyn11-1 was highly salt-tolerant, retaining more than 77.4% of activity in the presence of 0.25-4 M NaCl and displaying more than 47.2% relative activity after being incubated in the presence of 5 M NaCl at 37 °C for 10 min. In addition, 5 mM ß-Mercaptoethanol, Cu2+, Co2+, and Mn2+ increased the xylanase activity by 22.3%, 8.8%, 7.1%, and 4.4%, respectively. Significantly, 93.4% and 59.8% of the optimal activity was retained in the presence of 2% and 10% (v/v) ethanol, respectively. Under optimal conditions, the Km,Vmax, and Kcat value of Xyn11-1 for beechwood xylan were 3.7 mg ml-1, 101.0 µmol min-1 mg-1 and 42.1 s-1, respectively. Xyn11-1 is a strict endo-ß-1,4-xylanase, its main enzymatic products being xylotetraose and xylopentaose. Xyn11-1 is the first reported GH11 xylanase isolated from saline-alkali soil, and has excellent tolerance of high pH, high salt concentrations and ethanol, which indicates its great potential for basic research and industrial applications.