A long-term growth stable Halomonas sp. deleted with multiple transposases guided by its metabolic network model Halo-ecGEM.

Microbial instability is a common problem during bio-production based on microbial hosts. Halomonas bluephagenesis has been developed as a chassis for next generation industrial biotechnology (NGIB) under open and unsterile conditions. However, the hidden genomic information and peculiar metabolism have significantly hampered its deep exploitation for cell-factory engineering. Based on the freshly completed genome sequence of H. bluephagenesis TD01, which reveals 1889 biological process-associated genes grouped into 84 GO-slim terms. An enzyme constrained genome-scale metabolic model Halo-ecGEM was constructed, which showed strong ability to simulate fed-batch fermentations. A visible salt-stress responsive landscape was achieved by combining GO-slim term enrichment and CVT-based omics profiling, demonstrating that cells deploy most of the protein resources by force to support the essential activity of translation and protein metabolism when exposed to salt stress. Under the guidance of Halo-ecGEM, eight transposases were deleted, leading to a significantly enhanced stability for its growth and bioproduction of various polyhydroxyalkanoates (PHA) including 3-hydroxybutyrate (3HB) homopolymer PHB, 3HB and 3-hydroxyvalerate (3HV) copolymer PHBV, as well as 3HB and 4-hydroxyvalerate (4HB) copolymer P34HB. This study sheds new light on the metabolic characteristics and stress-response landscape of H. bluephagenesis, achieving for the first time to construct a long-term growth stable chassis for industrial applications. For the first time, it was demonstrated that genome encoded transposons are the reason for microbial instability during growth in flasks and fermentors.

Flux optimization using multiple promoters in Halomonas bluephagenesis as a model chassis of the next generation industrial biotechnology.

Predictability and robustness are challenges for bioproduction because of the unstable intracellular synthetic activities. With the deeper understanding of the gene expression process, fine-tuning has become a meaningful tool for biosynthesis optimization. This study characterized several gene expression elements and constructed a multiple inducible system that responds to ten different small chemical inducers in halophile bacterium Halomonas bluephagenesis. Genome insertion of regulators was conducted for the purpose of gene cluster stabilization and regulatory plasmid simplification. Additionally, dynamic ranges of the multiple inducible systems were tuned by promoter sequence mutations to achieve diverse scopes for high-resolution gene expression control. The multiple inducible system was successfully employed to precisely control chromoprotein expression, lycopene and poly-3-hydroxybutyrate (PHB) biosynthesis, resulting in colorful bacterial pictures, optimized cell growth, lycopene and PHB accumulation. This study demonstrates a desirable approach for fine-tuning of rational and efficient gene expressions, displaying the significance for metabolic pathway optimization.

Engineering low-salt growth Halomonas Bluephagenesis for cost-effective bioproduction combined with adaptive evolution.

Halophilic Halomonas bluephagenesis has been engineered to produce various added-value bio-compounds with reduced costs. However, the salt-stress regulatory mechanism remained unclear. H. bluephagenesis was randomly mutated to obtain low-salt growing mutants via atmospheric and room temperature plasma (ARTP). The resulted H. bluephagenesis TDH4A1B5 was constructed with the chromosomal integration of polyhydroxyalkanoates (PHA) synthesis operon phaCAB and deletion of phaP1 gene encoding PHA synthesis associated protein phasin, forming H. bluephagenesis TDH4A1B5P, which led to increased production of poly(3-hydroxybutyrate) (PHB) and poly(3-hydroxybutyrate-co-4-hydrobutyrate) (P34HB) by over 1.4-fold. H. bluephagenesis TDH4A1B5P also enhanced production of ectoine and threonine by 50% and 77%, respectively. A total 101 genes related to salinity tolerance was identified and verified via comparative genomic analysis among four ARTP mutated H. bluephagenesis strains. Recombinant H. bluephagenesis TDH4A1B5P was further engineered for PHA production utilizing sodium acetate or gluconate as sole carbon source. Over 33% cost reduction of PHA production could be achieved using recombinant H. bluephagenesis TDH4A1B5P. This study successfully developed a low-salt tolerant chassis H. bluephagenesis TDH4A1B5P and revealed salt-stress related genes of halophilic host strains.

Synthetic biology of extremophiles: a new wave of biomanufacturing.

Microbial biomanufacturing, powered by the advances of synthetic biology, has attracted growing interest for the production of diverse products. In contrast to conventional microbes, extremophiles have shown better performance for low-cost production owing to their outstanding growth and synthesis capacity under stress conditions, allowing unsterilized fermentation processes. We review increasing numbers of products already manufactured utilizing extremophiles in recent years. In addition, genetic parts, molecular tools, and manipulation approaches for extremophile engineering are also summarized, and challenges and opportunities are predicted for non-conventional chassis. Next-generation industrial biotechnology (NGIB) based on engineered extremophiles promises to simplify biomanufacturing processes and achieve open and continuous fermentation, without sterilization, and utilizing low-cost substrates, making NGIB an attractive green process for sustainable manufacturing.

Advances and trends in microbial production of polyhydroxyalkanoates and their building blocks.

With the rapid development of synthetic biology, a variety of biopolymers can be obtained by recombinant microorganisms. Polyhydroxyalkanoates (PHA) is one of the most popular one with promising material properties, such as biodegradability and biocompatibility against the petrol-based plastics. This study reviews the recent studies focusing on the microbial synthesis of PHA, including chassis engineering, pathways engineering for various substrates utilization and PHA monomer synthesis, and PHA synthase modification. In particular, advances in metabolic engineering of dominant workhorses, for example Halomonas, Ralstonia eutropha, Escherichia coli and Pseudomonas, with outstanding PHA accumulation capability, were summarized and discussed, providing a full landscape of diverse PHA biosynthesis. Meanwhile, we also introduced the recent efforts focusing on structural analysis and mutagenesis of PHA synthase, which significantly determines the polymerization activity of varied monomer structures and PHA molecular weight. Besides, perspectives and solutions were thus proposed for achieving scale-up PHA of low cost with customized material property in the coming future.

Engineering an oleic acid-induced system for Halomonas, E. coli and Pseudomonas.

Ligand-induced system plays an important role for microbial engineering due to its tunable gene expression control over timings and levels. An oleic acid (OA)-induced system was recently constructed based on protein FadR, a transcriptional regulator involved in fatty acids metabolism, for metabolic control in Escherichia coli. In this study, we constructed a synthetic FadR-based OA-induced systems in Halomonas bluephagenesis by hybridizing the porin promoter core region and FadR-binding operator (fadO). The dynamic control range was optimized over 150-fold, and expression leakage was significantly reduced by tuning FadR expression and positioning fadO, forming a series of OA-induced systems with various expression strengths, respectively. Additionally, ligand orthogonality and cross-species portability were also studied and showed highly linear correlation among Halomonas spp., Escherichia coli and Pseudomonas spp. Finally, OA-induced systems with medium- and small-dynamic control ranges were employed to dynamically control the expression levels of morphology associated gene minCD, and monomer precursor 4-hydroxybutyrate-CoA (4HB-CoA) synthesis pathway for polyhydroxyalkanoates (PHA), respectively, in the presence of oleic acid as an inducer. As a result, over 10 g/L of poly-3-hydroxybutyrate (PHB) accumulated by elongated cell sizes, and 6 g/L of P(3HB-co-9.57 mol% 4HB) were obtained by controlling the dose and induction time of oleic acid only. This study provides a systematic approach for ligand-induced system engineering, and demonstrates an alternative genetic tool for dynamic control of industrial biotechnology.

Effective production of Poly(3-hydroxybutyrate-co-4-hydroxybutyrate) by engineered Halomonas bluephagenesis grown on glucose and 1,4-Butanediol.

Halomonas bluephagenesis has been engineered to produce flexible copolymers P34HB or poly(3-hydroxybutyrate-co-4-hydroxybutyrate) from glucose and petrol-chemical precursor, γ-butyrolactone. Herein, gene cluster aldD-dhaT was constructed in recombinant H. bluephagenesis for catalyzing 1,4-butanediol (BDO) into 4-hydroxybutyrate, which could grow to 86 g L-1 dry cell mass (DCM) containing 77 wt% P(3HB-co-14 mol% 4HB) in 7-L bioreactor fed with glucose and bio-based BDO. Furthermore, 4HB monomer ratio could be increased to 16 mol% by engineered H. bluephagenesis TDH4-WZY254 with defected outer-membrane. Upon deletion of 4HB degradation pathway, followed by aldD-dhaT integration, the resulted H. bluephagenesis TDB141ΔAC was grown to 95 g L-1 DCM containing 79 wt% P(3HB-co-14 mol% 4HB) with a BDO conversion efficiency of 86% under fed-batch fermentation. Notably, 4HB molar ratio can be significantly improved to 21 mol% with negligible effects on cell growth and P34HB synthesis by adding 50% more BDO. This study successfully demonstrated a fully bio-based P34HB effectively produced by H. bluephagenesis.

Long non-coding RNA GAS6-AS1 enhances breast cancer cell aggressiveness by functioning as a competing endogenous RNA of microRNA-215-5p to enhance SOX9 expression.

Long non-coding (lnc) RNAs play crucial functions in human cancer. However, until recently, the involvement of the lncRNA GAS6-AS1 in breast cancer (BCa) malignancy has not been studied exhaustively. The roles and underlying mode of action of GAS6-AS1 action in BCa progression were examined through functional experiments. A decline in GAS6-AS1 level led to a significant decrease in BCa cell proliferation, and the ability for colony formation. Here, GAS6-AS1 competed as endogenous RNA by sequestering microRNA-215-5p (miR-215-5p) causing an enhanced expression of SRY-box transcription factor 9 (SOX9). The effects of silencing GAS6-AS1 on BCa malignant phenotypes could be ameliorated by inhibiting miR-215-5p or restoring SOX9. Thus, GAS6-AS1 acted as a lncRNA that drives tumor in BCa, and enabled progression of BCa through miR-215-5p /SOX9 axis regulation. These outcomes show that the GAS6-AS1/miR-215-5p/SOX9 axis is a potentially effective target for cancer treatment and management.

Reversible thermal regulation for bifunctional dynamic control of gene expression in Escherichia coli.

Genetically programmed circuits allowing bifunctional dynamic regulation of enzyme expression have far-reaching significances for various bio-manufactural purposes. However, building a bio-switch with a post log-phase response and reversibility during scale-up bioprocesses is still a challenge in metabolic engineering due to the lack of robustness. Here, we report a robust thermosensitive bio-switch that enables stringent bidirectional control of gene expression over time and levels in living cells. Based on the bio-switch, we obtain tree ring-like colonies with spatially distributed patterns and transformer cells shifting among spherical-, rod- and fiber-shapes of the engineered Escherichia coli. Moreover, fed-batch fermentations of recombinant E. coli are conducted to obtain ordered assembly of tailor-made biopolymers polyhydroxyalkanoates including diblock- and random-copolymer, composed of 3-hydroxybutyrate and 4-hydroxybutyrate with controllable monomer molar fraction. This study demonstrates the possibility of well-organized, chemosynthesis-like block polymerization on a molecular scale by reprogrammed microbes, exemplifying the versatility of thermo-response control for various practical uses.

Engineering Halomonas bluephagenesis as a chassis for bioproduction from starch.

Halomonas bluephagenesis has been successfully engineered to produce multiple products under open unsterile conditions utilizing costly glucose as the carbon source. It would be highly interesting to investigate if H. bluephagenesis, a chassis for the Next Generation Industrial Biotechnology (NGIB), can be reconstructed to become an extracellular hydrolytic enzyme producer replacing traditional enzyme producer Bacillus spp. If successful, cost of bulk hydrolytic enzymes such as amylase and protease, can be significantly reduced due to the contamination resistant and robust growth of H. bluephagenesis. This also allows H. bluephagenesis to be able to grow on low cost substrates such as starch. The modularized secretion machinery was constructed and fine-tuned in H. bluephagenesis using codon-optimized gene encoding α-amylase from Bacillus lichenifomis. Screening of suitable signal peptides and linkers based on super-fold green fluorescence protein (sfGFP) for enhanced expression in H. bluephagenesis resulted in a 7-fold enhancement of sfGFP secretion in the recombinant H. bluephagenesis. When the gene encoding sfGFP was replaced by α-amylase encoding gene, recombinant H. bluephagenesis harboring this amylase secretory system was able to produce poly(3-hydroxybutyrate) (PHB), poly(3-hydroxybutyrate-co-4-hydroxybutyrate) (P34HB), poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV), ectoine and L-threonine utilizing starch as the growth substrate, respectively. Recombinant H. bluephagenesis TN04 expressing genes encoding α-amylase and glucosidase on chromosome and plasmid-based systems, respectively, was able to grow on corn starch to approximately 10 g/L cell dry weight containing 51% PHB when grown in shake flasks. H. bluephagenesis was demonstrated to be a chassis for productions of extracellular enzymes and multiple products from low cost corn starch.

Synthetic Biology and Genome-Editing Tools for Improving PHA Metabolic Engineering.

Polyhydroxyalkanoates (PHAs) are a diverse family of biopolyesters synthesized by many natural or engineered bacteria. Synthetic biology and DNA-editing approaches have been adopted to engineer cells for more efficient PHA production. Recent advances in synthetic biology applied to improve PHA biosynthesis include ribosome-binding site (RBS) optimization, promoter engineering, chromosomal integration, cell morphology engineering, cell growth behavior reprograming, and downstream processing. More importantly, the genome-editing tool clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) has been applied to optimize the PHA synthetic pathway, regulate PHA synthesis-related metabolic flux, and control cell shapes in model organisms, such as Escherichia coli, and non-model organisms, such as Halomonas. These synthetic biology methods and genome-editing tools contribute to controllable PHA molecular weights and compositions, enhanced PHA accumulation, and easy downstream processing.

Siomycin A Induces Apoptosis in Human Lung Adenocarcinoma A549 Cells by Suppressing the Expression of FoxM1.

Forkhead box M1 (FoxM1), a transcription factor of the Forkhead family, is demonstrated to be critical for proliferation, apoptosis, migration and invasion of lung cancer. In this study, we extensively investigated the anticancer effect of siomycin A, which was identified as an inhibitor of FoxM1 transcriptional activity, on human lung adenocarcinoma A549 cells. Our study indicated that treatment with siomycin A resulted in the suppression of FoxM1 expression, which consequently contributed to its effect of cell growth inhibition and cell apoptosis induction in A549 cells. Then the molecular mechanism of siomycin A's apoptotic action on A549 cells was further investigated. The results revealed that siomycin A induced apoptosis by influencing the downstream events of FoxM1, including inhibiting the expression of Bcl-2 and Mcl-1, as well as leading to caspase-3 cleavage. Taken together, our findings may be useful for understanding the mechanism of action of siomycin A on lung cancer cells and provide new insights into the possible application of such a compound in lung cancer therapy in the future.

Genetic dynamic analysis of the influenza A H5N1 NS1 gene in China.

The direct precursors of the A/Goose/Guangdong/1/1996 (GS/GD) virus lineage and its reassortants have been established geographically and ecologically. To investigate the variation and evolutionary dynamics of H5N1 viruses, whole-genome viral sequences (n = 164) were retrieved from the NCBI Influenza Virus Resource. Here, we present phylogenetic evidence for intrasubtype reassortments among H5N1 viruses isolated from China during 1996-2012. On the basis of phylogenetic analysis, we identified four major groups and further classified the reassortant viruses into three subgroups. Putative mosaic structures were mostly found in the viral ribonucleoprotein (vRNP) complexes and 91.0% (10/11) mosaics were obtained from terrestrial birds. Sequence variability and selection pressure analyses revealed that both surface glycoproteins (HA and NA) and nonstructural protein 1 (NS1) have higher dN/dS ratio and variability than other internal proteins. Furthermore, we detected 47 positively selected sites in genomic segments with the exception of PB2 and M1 genes. Hemagglutinin (HA) and neuraminidase (NA) are considered highly variable due to host immune pressure, however, it is not known what drives NS1 variability. Therefore, we performed a thorough analysis of the genetic variation and selective pressure of NS1 protein (462 available NS1 sequences). We found that most of positively selected sites and variable amino acids were located in the C-terminal effector domain (ED) of NS1. In addition, we focused on the NS1-RNA and NS1-protein interactions that were involved in viral replication mechanisms and host immune response. Transcriptomic analysis of H5N1-infected monkey lungs showed that certain PI3K-related genes were up-regulated.

Argonaute protein as a linker to command center of physiological processes.

MicroRNAs (miRNAs) post-transcriptionally regulate gene expression by binding to target mRNAs with perfect or imperfect complementarity, recruiting an Argonaute (AGO) protein complex that usually results in degradation or translational repression of the target mRNA. AGO proteins function as the Slicer enzyme in miRNA and small interfering RNA (siRNA) pathways involved in human physiological and pathophysiological processes, such as antiviral responses and disease formation. Although the past decade has witnessed rapid advancement in studies of AGO protein functions, to further elucidate the molecular mechanism of AGO proteins in cellular function and biochemical process is really a challenging area for researchers. In order to understand the molecular causes underlying the pathological processes, we mainly focus on five fundamental problems of AGO proteins, including evolution, functional domain, subcellular location, post-translational modification and protein-protein interactions. Our discussion highlight their roles in early diagnosis, disease prevention, drug target identification, drug response, etc.

Genome-wide analysis of bZIP-encoding genes in maize.

In plants, basic leucine zipper (bZIP) proteins regulate numerous biological processes such as seed maturation, flower and vascular development, stress signalling and pathogen defence. We have carried out a genome-wide identification and analysis of 125 bZIP genes that exist in the maize genome, encoding 170 distinct bZIP proteins. This family can be divided into 11 groups according to the phylogenetic relationship among the maize bZIP proteins and those in Arabidopsis and rice. Six kinds of intron patterns (a-f) within the basic and hinge regions are defined. The additional conserved motifs have been identified and present the group specificity. Detailed three-dimensional structure analysis has been done to display the sequence conservation and potential distribution of the bZIP domain. Further, we predict the DNA-binding pattern and the dimerization property on the basis of the characteristic features in the basic and hinge regions and the leucine zipper, respectively, which supports our classification greatly and helps to classify 26 distinct subfamilies. The chromosome distribution and the genetic analysis reveal that 58 ZmbZIP genes are located in the segmental duplicate regions in the maize genome, suggesting that the segment chromosomal duplications contribute greatly to the expansion of the maize bZIP family. Across the 60 different developmental stages of 11 organs, three apparent clusters formed represent three kinds of different expression patterns among the ZmbZIP gene family in maize development. A similar but slightly different expression pattern of bZIPs in two inbred lines displays that 22 detected ZmbZIP genes might be involved in drought stress. Thirteen pairs and 143 pairs of ZmbZIP genes show strongly negative and positive correlations in the four distinct fungal infections, respectively, based on the expression profile and Pearson's correlation coefficient analysis.

[Effects of silicon carbide on the cure depth, hardness and compressive strength of composite resin].

The hardness, compressive strength and cure depth are important indices of the composite resin. This investigation was made with regard to the effects of silicon carbide on the cure depth, hardness and compressive strength of the light-curing composite resin. Different amounts of silicon carbide were added to the light-curing composite resin, which accounted for 0 wt%, 1 wt%, 0.6 wt%, 0.3 wt%, 0.1 wt%, 0.05 wt% and 0.005 wt% of the composite resin, respectively. The hardness, compressive strength and cure depth of the six afore-mentioned groups of composite resin were measured by the vernier caliper, the vickers hardness tester and the tensile strength of machine, respectively. The results showed that silicon carbide improved the hardness and compressive strength of the light-curing composite resin,when the concentration was 0.05 wt%. And the cure depth was close to that of control.

[Effects of diaryliodonium on the properties of light-curing composite resin].

OBJECTIVE: To test the effects of two kinds of diaryliodonium on the degree of conversion (DC), the compressive strength (CS) and the cure depth (CD) of the light-curing composite resin. METHODS: BisS-GMA was used as reisin matrix, with TEGDMA as diluent and SiO2 as inorganic filler. CQ, DMAEMA and two different kinds of diaryliodonium (diphenyliodonium hexafluorophosphate and bis (p-tolyl) iodonium hexafluorophosphate) were used as photoinitiator, respectively. The DC, CS, CD were measured and compared by the FTIR spectroscopy, and the tensile strength of machine et al. RESULTS: Diaryliodonium led to greater DC, CS, and CD of the light-curing composite resin. The DC, CS, and CD of the light-curing composite resin increased more with bis (p-tolyl) iodonium hexafluorophosphate than with diphenyliodonium hexafluorophosphate. CONCLUSION: Diaryliodonium can improve the DC, CS, and CD properties of light-curing composite resin. Bis (p-tolyl) iodonium hexafluorophosphate has better performance than diphenyliodonium hexafluorophosphate in improving photosensibility and solubility of the photoinitiator system.

[Effects of diphenyliodonium on the degree of conversion and the compressive strength of composite resin].

The degree of conversion (DC) and the compressive strength (CS) are important indexes of composite resin. This study is aimed to investigate the effects of diphenyliodonium on the DC and the CS of the light-curing composite resin. Different amount of diphenyliodonium hexaflourophosphate was added to the light-curing composite resin; the concentrations of diphenyliodonium hexaflourophosphate were 0 wt%, 0.5 wt%, 1.0 wt%, 2.0 wt%, 3.0 wt% and 4.0 wt% of the reisin matrix (containing BisS-GMA and TEGDMA) respectively. The DC and the CS of the six aforementioned groups of the composite resin were measured by the Fourier transform infrared spectroscopy and the tensile strength of machine, respectively. Diphenyliodonium hexaflourophosphate can improve the DC and CS of the light-curing composite resin. When the concentration is 2.0 wt%, the DC and CS can achieve better performance.