Comparison of gal-lac operons in wild-type galactose-positive and -negative Streptococcus thermophilus by genomics and transcription analysis.

Streptococcus thermophilus is one of the most important homo-fermentative thermophilic bacteria, which is widely used as a starter culture in dairy industry. Both wild-type galactose-negative (Gal-) S. thermophilus AR333 and galactose-positive (Gal+) S. thermophilus S-3 in this study were isolated from Chinese traditional dairy products. Here, to access the mechanism of the difference of galactose utilization between strains AR333 and S-3, the expression of gal-lac operons was examined using real-time qPCR in the presence of different sugars, and the gene organization of gal-lac operons was characterized using comparative genomics analysis. As compared with medium containing glucose, the expression of gal-lac operons in AR333 and S-3 was significantly activated (> 5-fold) in the presence of galactose or lactose in the medium. More importantly, the expression of gal operon in S-3 was higher than that of AR333, suggesting that the strength of gal promoter in AR333 and S-3 may be different. The genomes of AR333 and S-3 were the first time sequenced to provide insight into the difference of gal-lac operons in these two strains. Comparative genomics analysis showed that gene order and individual gene size of gal-lac operons are conserved in AR333 and S-3. The DNA sequence of gal operon responsible for galactose utilization between AR333 and S-3 is almost identical except that galK promoter of S-3 possesses single base pair mutation (G to A substitution) at -9 box galK region. Moreover, the expression of red fluorescent protein can be activated by galK promoter of S-3, but cannot by galK promoter of AR333 in galactose medium, suggesting that gal operon is silent in AR333 and active in S-3 under galactose-containing medium. Overall, our results indicated that single point mutation at -9 box in the galK promoter can significantly affect the expression of gal operon and is largely responsible for the Gal+ phenotype of S. thermophilus.

Synbiological systems for complex natural products biosynthesis.

Natural products (NPs) continue to play a pivotal role in drug discovery programs. The rapid development of synthetic biology has conferred the strategies of NPs production. Synthetic biology is a new engineering discipline that aims to produce desirable products by rationally programming the biological parts and manipulating the pathways. However, there is still a challenge for integrating a heterologous pathway in chassis cells for overproduction purpose due to the limited characterized parts, modules incompatibility, and cell tolerance towards product. Enormous endeavors have been taken for mentioned issues. Herein, in this review, the progresses in naturally discovering novel biological parts and rational design of synthetic biological parts are reviewed, combining with the advanced assembly technologies, pathway engineering, and pathway optimization in global network guidance. The future perspectives are also presented.

Construction of polyketide overproducing Escherichia coli strains via synthetic antisense RNAs based on in silico fluxome analysis and comparative transcriptome analysis.

Rapid assessment and optimization of the incompatible metabolic modules remain a challenge. Here, we developed a systematic approach to characterize the module interactions and improve the problematic modules during the 6-deoxyerythronolide B (6dEB) biosynthesis in E. coli. Tremendous differences in the overall trends of flux changes of various metabolic modules were firstly uncovered based on in silico fluxome analysis and comparative transcriptome analysis. Potential targets for improving 6dEB biosynthesis were identified through analyzing these discrepancies. All 25 predicted targets at modules of PP pathway and nucleotide metabolism were firstly tested for improving the 6dEB production in E. coli via synthetic antisense RNAs. Down-regulation of 18 targets genes leads to more than 20% increase in 6dEB yield. Combinatorial repression of targets with greater than 60% increase in 6dEB titer, e.g., anti-guaB/anti-zwf led to a 296.2% increase in 6dEB production (210.4 mg/L in flask) compared to the control (53.1 mg/L). This is the highest yield yet reported for polyketide heterologous biosynthesis in E. coli. This study demonstrates a strategy to enhance the yield of heterologous products in the chassis cell and indicates the effectiveness of antisense RNA for use in metabolic engineering.

Identification of novel knockout targets for improving terpenoids biosynthesis in Saccharomyces cerevisiae.

Many terpenoids have important pharmacological activity and commercial value; however, application of these terpenoids is often limited by problems associated with the production of sufficient amounts of these molecules. The use of Saccharomyces cerevisiae (S. cerevisiae) for the production of heterologous terpenoids has achieved some success. The objective of this study was to identify S. cerevisiae knockout targets for improving the synthesis of heterologous terpeniods. On the basis of computational analysis of the S. cerevisiae metabolic network, we identified the knockout sites with the potential to promote terpenoid production and the corresponding single mutant was constructed by molecular manipulations. The growth rates of these strains were measured and the results indicated that the gene deletion had no adverse effects. Using the expression of amorphadiene biosynthesis as a testing model, the gene deletion was assessed for its effect on the production of exogenous terpenoids. The results showed that the dysfunction of most genes led to increased production of amorphadiene. The yield of amorphadiene produced by most single mutants was 8-10-fold greater compared to the wild type, indicating that the knockout sites can be engineered to promote the synthesis of exogenous terpenoids.

[Distribution of refraction and ocular biometric parameters in a population of junior middle school children in Anyang of Henan province].

OBJECTIVE: To investigate the distribution of spherical equivalent and ocular biometric parameters in a population of grade 7 children in central China. METHODS: 2 363 grade 7 students of junior high schools were randomly sampled. The students have been examined at baseline and followed up annually. Detailed questionnaires and most of the ocular examinations were performed. Spherical equivalent (Seq) was calculated as sphere +1/2 cylinder from cycloplegic autorefraction. Myopia was defined as spherical equivalent ≤ -0.5D, hyperopia as spherical equivalent ≥ +0.5D and emmetropia between -0.5D and +0.5D. The Lensar LS900 was used to measure corneal curvatures, axial length and anterior chamber depth. Right eyes results were analyzed. RESULTS: Among 2 267 children who have the baseline examination, 1 839 children participated the follow up examination(response rate, 81.1%), with an mean age of 14.7 years (range, 12.9-17.6) and male ratio of 48.4%. The mean spherical equivalent refraction was (-2.62 ± 2.19)D. The prevalence rates of myopia, high myopia, emmetropia and hyperopia were 82.7%, 7.1%, 9.8% and 7.5%. The cumulative incidence rates of myopia and high myopia were 47.1% and 4.5% respectively. Axial length, anterior chamber depth, and corneal curvature were normally distributed. The mean axial length, anterior chamber depth, and corneal curvature were (24.8 ± 1.1)mm, (3.18 ± 0.25)mm, (42.8 ± 1.4)D and (43.9 ± 1.6)D respectively. Axial length was longer, anterior chamber depth was deeper, and corneal were flatter in the male(P = 0.000). Axial length and anterior chamber depth correlated negatively with refraction. CONCLUSIONS: A moderate myopic distribution of refraction was present in the grade 9 students in central China. The prevalence rates of myopia and high myopia were relatively high in this 14-year-old population compared to other countries. It is necessary to strengthen the prevention of juvenile myopia.

Identification of novel knockout and up-regulated targets for improving isoprenoid production in E. coli.

Discovery of novel potential genetic targets to increase the supply of isoprenoid precursors, isopentyl/dimethylallyl diphosphate, is of importance for microbial production of isoprenoids. Here, to improve isoprenoid precursor supply, a flux distribution comparison analysis, based on the genome-scale model, was utilized to simultaneously predict the knockout, down- and up-regulated targets in Escherichia coli. 51 targets were in silico discovered. All knockout and up-regulated targets were experimentally tested to enhance lycopene production. Five knockout targets (deoB, yhfw, yahI, pta and eutD) and four up-regulated targets (ompN, ompE, ndk and cmk) led to 10-45% increases of lycopene yield, respectively, which had not been uncovered in previous studies. When engineering of the five most significant targets gdhA, eutD, tpiA, ompE and ompN, were combined the lycopene titer improved by 174% in shake-flask and 81% in bioreactor fermentations with a maximum yield of 454 mg l(-1).

ABCC5, a gene that influences the anterior chamber depth, is associated with primary angle closure glaucoma.

Anterior chamber depth (ACD) is a key anatomical risk factor for primary angle closure glaucoma (PACG). We conducted a genome-wide association study (GWAS) on ACD to discover novel genes for PACG on a total of 5,308 population-based individuals of Asian descent. Genome-wide significant association was observed at a sequence variant within ABCC5 (rs1401999; per-allele effect size =  -0.045 mm, P = 8.17 × 10(-9)). This locus was associated with an increase in risk of PACG in a separate case-control study of 4,276 PACG cases and 18,801 controls (per-allele OR = 1.13 [95% CI: 1.06-1.22], P = 0.00046). The association was strengthened when a sub-group of controls with open angles were included in the analysis (per-allele OR = 1.30, P = 7.45 × 10(-9); 3,458 cases vs. 3,831 controls). Our findings suggest that the increase in PACG risk could in part be mediated by genetic sequence variants influencing anterior chamber dimensions.

[Design and construction of artificial biological systems for complex natural products biosynthesis].

Natural products (NPs) are important drug pools for human disease prevention and treatment. The great advances in synthetic biology have greatly revolutionized the strategies of NPs development and production. This review entitled with design and construction of artificial biological systems for complex NPs biosynthesis, mainly introduced the progresses in artificial design of synthetic biological parts, naturally mining novel synthetic parts of NPs, the assembly & adaption of the artificial biological modules & systems.

Quantitative design of regulatory elements based on high-precision strength prediction using artificial neural network.

Accurate and controllable regulatory elements such as promoters and ribosome binding sites (RBSs) are indispensable tools to quantitatively regulate gene expression for rational pathway engineering. Therefore, de novo designing regulatory elements is brought back to the forefront of synthetic biology research. Here we developed a quantitative design method for regulatory elements based on strength prediction using artificial neural network (ANN). One hundred mutated Trc promoter & RBS sequences, which were finely characterized with a strength distribution from 0 to 3.559 (relative to the strength of the original sequence which was defined as 1), were used for model training and test. A precise strength prediction model, NET90_19_576, was finally constructed with high regression correlation coefficients of 0.98 for both model training and test. Sixteen artificial elements were in silico designed using this model. All of them were proved to have good consistency between the measured strength and our desired strength. The functional reliability of the designed elements was validated in two different genetic contexts. The designed parts were successfully utilized to improve the expression of BmK1 peptide toxin and fine-tune deoxy-xylulose phosphate pathway in Escherichia coli. Our results demonstrate that the methodology based on ANN model can de novo and quantitatively design regulatory elements with desired strengths, which are of great importance for synthetic biology applications.

Synthetic biology triggers new era of antibiotics development.

As a discipline to design and construct organisms with desired properties, synthetic biology has generated rapid progresses in the last decade. Combined synthetic biology with the traditional process, a new universal workflow for drug development has been becoming more and more attractive. The new methodology exhibits more efficient and inexpensive comparing to traditional methods in every aspect, such as new compounds discovery & screening, process design & drug manufacturing. This article reviews the application of synthetic biology in antibiotics development, including new drug discovery and screening, combinatorial biosynthesis to generate more analogues and heterologous expression of biosynthetic gene clusters with systematic engineering the recombinant microbial systems for large scale production.

Genome-wide association analyses identify three new susceptibility loci for primary angle closure glaucoma.

Primary angle closure glaucoma (PACG) is a major cause of blindness worldwide. We conducted a genome-wide association study including 1,854 PACG cases and 9,608 controls across 5 sample collections in Asia. Replication experiments were conducted in 1,917 PACG cases and 8,943 controls collected from a further 6 sample collections. We report significant associations at three new loci: rs11024102 in PLEKHA7 (per-allele odds ratio (OR)=1.22; P=5.33×10(-12)), rs3753841 in COL11A1 (per-allele OR=1.20; P=9.22×10(-10)) and rs1015213 located between PCMTD1 and ST18 on chromosome 8q (per-allele OR=1.50; P=3.29×10(-9)). Our findings, accumulated across these independent worldwide collections, suggest possible mechanisms explaining the pathogenesis of PACG.

[Assess correlation between bleb morphology at long-term intraocular pressure effect in primary angle-closure glaucoma following trabeculectomy].

OBJECTIVE: To evaluate the correlation between morphologic appearance of blebs at 3 month and long-term intraocular pressure (IOP) effect in patients with primary angle-closure glaucoma (PACG) after trabeculectomy. METHODS: Multi-centered cases series. Data were collected from 176 patients aged ≥ 40 years with PACG who were participated in a randomized clinical trial that aimed at addressing the efficacy of augmented releasable sutures after trabeculectomy. The bleb morphology was graded using the Modified Indian Bleb Appearance Grading Scale (IBAGS) based on standard photos at 3 month after trabeculectomy. IOPs were measured with Goldmann applanation tonometer. The correlation between bleb components and other selected testing influencing factors and long-term IOP was tested by linear Logistic regression analysis. RESULTS: 150 patients (85.7%) completed 18 months of follow up. IOP was (15.6 ± 5.4) mm Hg at 18 month of post-operation. 135 eyes had an IOP ≤ 21 mm Hg without additional medications, 10 eyes ≥ 21 mm Hg, and the remaining 5 eyes required one or two medications to maintain normal IOP. Using IBAGS system, bleb was graded in 142 eyes as follows: H(0) in 3 eyes, H(1) in 45 eyes, H(2) in 90 eyes, and H(3) in 4 eyes, while V(0) was observed in 66 eyes, V(1-3) in 76 eyes. IOP at 18 months in bleb with microcysts was 2.77 mm Hg lower (ß = -2.77, 95%CI = -0.46 to -5.08) than those without microcysts and in bleb with non-vascular was 2.07 mm Hg lower (ß = -2.07, 95%CI = -0.15 to -3.98) than those with vascular at 3 months after surgery. IOP was significantly (ß = -1.20, 95%CI: -0.00 to -2.40) decreased by 1.2 mm Hg with 10 years of age increase (P < 0.05). CONCLUSIONS: Early filtering bleb with microcysts, vascular, and age are identified as important factors to predict long-term IOP effect in patients with PACG after trabeculectomy but not early morphological appearance of filtering bleb.

Evaluation of LOXL1 polymorphisms in exfoliation syndrome in a Chinese population.

PURPOSE: To evaluate the association profiles of the lysyl oxidase-like 1 (LOXL1) gene polymorphisms with exfoliation syndrome in a Chinese population. METHODS: Fifty unrelated patients with exfoliation syndrome and 125 control subjects were included. Genotypes of the three single nucleotide polymorphisms (SNPs) of LOXL1 (rs1048661, rs3825942, and rs2165241) were analyzed by direct sequencing, and a case-control association study was performed. RESULTS: The three SNPs were significantly associated with exfoliation syndrome (XFS) and exfoliation glaucoma (XFG) individually. After controlling for rs3825942 and rs2165241, the association between rs1048661 and XFS/XFG remained significant (p=3.6 x 10(-7)). At this SNP, the T allele and TT genotype conferred a 7.59-(95% confidence interval [CI]: 3.87-14.89, p=6.95 x 10(-11)) and 8.69-(95% CI: 4.15-18.20, p<1.00 x 10(-7)) fold increased risk to the disease. The alleles of T at rs1048661 and C at rs2165241 were found to be risk alleles in Chinese subjects, which were opposite to Caucasian individuals. The haplotypes T-G, defined by SNPs rs1048661 and rs3825942, and T-C by SNPs rs1048661 and rs2165241, were also significantly associated with the disorder. However when the genotypic or allelic frequencies of the three SNPs were compared between XFS and XFG, no significant difference was detected. CONCLUSIONS: LOXL1 is a susceptibility gene of XFS/XFG in the Chinese population, and the association is mainly attributed to SNP rs1048661. The risk alleles of rs1048661 and rs2165241 in Chinese subjects were found to be opposite to that of Caucasians. The genotypic and allelic distributions of these SNPs are similar between XFS and XFG.