Generation of high rapamycin producing strain via rational metabolic pathway‐based mutagenesis and further titer improvement with fed‐batch bioprocess optimization

Rapamycin is a triene macrolide antibiotic produced by Streptomyces hygroscopicus. Besides its wide application as an effective immunosuppressive agent, other important bioactivities have made rapamycin a potential drug lead for novel pharmaceutical development. However, the low titer of rapamycin in the original producer strain limits further industrialization efforts and restricts its use for other applications. Predicated on knowledge of the metabolic pathways related to rapamycin biosynthesis in S. hygroscopicus, we have rationally designed approaches to generate a rapamycin high producer strain of S. hygroscopicus HD‐04‐S. These have included alleviation of glucose repression, improved tolerance towards lysine and shikimic acid, and auxotrophy of tryptophan and phenylalanine through the application of stepwise UV mutagenesis. The resultant strain produced rapamycin at 450 mg/L in the shake flask scale. These fermentations were further scaled up in 120 and 20,000 L fermentors, respectively, at the pilot plant. Selected fermentation factors including agitation speed, pH, and on‐line supplementation were systematically evaluated. A fed‐batch strategy was established to maximize rapamycin production. With these efforts, an optimized fermentation process in the larger scale fermentor was developed. The final titer of rapamycin was 812 mg/L in the 120 L fermentor and 783 mg/L in the 20,000 L fermentor. This work highlights a high rapamycin producing strain derived by mutagenesis and subsequent screening, fermentation optimization of which has now made it feasible to produce rapamycin on an industrial scale by fermentation. The strategies developed here should also be applicable to titer improvement of other important microbial natural products on an industrial scale. Biotechnol. Bioeng. 2010;107: 506–515. © 2010 Wiley Periodicals, Inc.     

Genetic Dissection of Wheat Kernel Hardness Using Conditional QTL Mapping of Kernel Size and Protein-Related Traits

Kernel hardness (KH) is one of the primary quality parameters for common wheat (Triticum aestivum L.) and has a major impact on milling, flour quality, and end-product properties. In addition to Puroindoline (Pin) mutations and differences in Pin expression, other factors, such as kernel size and protein-related traits, play noticeable roles in determining hardness, but at the quantitative trait locus (QTL) level, the influence of these factors remains unclear. In this study, genetic relationships between KH and kernel size traits and between KH and protein-related traits were demonstrated by unconditional and conditional mapping using a wheat 90K genotyping assay with a segregating population of 173 recombinant inbred lines in four environments. Eight additive QTL for KH were detected using unconditional QTL mapping analysis; these QTL were primarily distributed on chromosomes 4B, 5A, 5B, and 6D, with phenotypic variation that ranged from 0.2 to 17.7%. In addition, one pair of epistatic QTL (QKH3B.4-65/QKH4B.6-2) was identified by unconditional mapping, and this pair accounted for 1.6% of the phenotypic variation. Through conditional mapping, after excluding the influences of kernel size and protein-related traits, 14 QTL were discovered and accounted for 0.6–18.5% of the phenotypic variation. Of them, the stable QTL QKH4B.4-17 made the largest contribution, which was partially contributed by the kernel length (KL), kernel thickness (KT), and dry gluten content (DGC). Furthermore, QKH4B.4-17 was crucially contributed by the kernel width (KW), kernel diameter (KD), kernel protein content (KPC), and wet gluten content (WGC) and was independent of the sedimentation volume (SV) and gluten index (GI). Another major QTL, QKH5B.10-63, was independent of the KW and KT; partly due to the variations in KL, KD, DGC, and WGC; and conclusively contributed by the KPC, SV, and GI. Seven additional QTL were only detected in the conditional analysis and were crucially contributed by kernel size or protein-related traits. These results demonstrated that kernel size and protein-related traits play significant roles in determining KH. The present study increases the understanding of the relationships between KH and kernel size and between KH and protein-related traits at the QTL level.     

编制校本实验报告 提高初中实验教学有效性

初中生物学实验课上容易出现无序和低效等问题,教师将自编学生实验或活动报告作为实验教学组织的一项措施,有效地提高了实验教学质量。     

G(o) controls the hyperpolarization-activated current in embryonic stem cell-derived cardiocytes

Hyperpolarization current (I(f)) is an important player in controlling heart rate and is stimulated by cAMP and inhibited by members of the pertussis toxin-sensitive G-protein G(i)/G(o) family. We have successfully derived cardiocytes from embryonic stem cells lacking G(o) or G(i2) and G(i3). We have established that both basal and isoproterenol-stimulated activities of I(f) in these cardiocytes have typical nodal-atrial characteristics and are unaffected by targeted gene inactivation of the G proteins G(o) or G(i2) and G(i3). Under basal conditions, both G(o) and G(i) are required for muscarinic inhibition of I(f) activity via a mechanism that involves the generation of nitric oxide, whereas, with prior stimulation by beta-agonists, only G(o) is required and G(i) and nitric oxide production are not. Our findings establish an essential role for G(o) in the antiadrenergic effect of muscarinic agent on I(f).     

Synthesis and antidiabetic activity of 5,7-dihydroxyflavonoids and analogs

In a study to evaluate the structural elements essential for the antidiabetic activity of flavonoids, we synthesized two series of flavonoids, 5,7-dihydroxyflavanones and 5,7-dihydroxyflavones. In a screening for potential antidiabetic activity, most of the flavonoids showed a remarkable in vitro activity, and compounds 1f, 2d, and 3c were significantly more effective than the positive control, metformin. The biological activity was mainly affected by structural modification at the ring B moiety of the flavonoid skeleton. The results suggest that 5,7-dihydroxyflavonoids can be considered as promising candidates in the development of new antidiabetic lead compounds.     

Genetic analysis and gene mapping of a rice few-tillering mutant in early backcross populations ( Oryza sativa L.)

A rice mutant,G069, characteristic of few tiller numbers, was found in anther culture progeny from theF ₁ hybrid between anindica-japonica cross, Gui630×02428. The mutant has another two major features: delayed tillering development and yellowing apex and margin on the mature leaves. As a donor parent,G069 was further backcrossed with the recurrent parent,02428, for two turns to develop aBC ₂F₂ population. Genetic analysis in theBC ₂F₂ population showed that the traits of few-tillering and yellowing apex and margin on the mature leaves were controlled by one recessive gene. A pool of equally mixed genomic DNA, from few-tillering individual plants inBC ₂F₂, was constructed to screen polymorphism with simple sequence repeat (SSR) markers in comparison with the02428 genome. One SSR marker and three restriction fragment length polymorphism (RFLP) markers were found possibly linked with the recessive gene. By using these markers, the gene of few-tillering was mapped on chromosome 2 between RFLP marker C424 and S13984 with a genetic distance of 2.4 cM and 0.6 cM, respectively. The gene is designatedft1.     

Population genetics of wild Hizikia fusiformis (Sargassaceae, Phaeophyta) along China’s coast

Hizikia fusiformis is one of the important commercially cultivated seaweeds in China. Inter-simple sequence repeats (ISSR) and sequence-related amplified polymorphism (SRAP) markers were used to assess genetic structure of nine wild H. fusiformis populations collected along the coast of China. Of the 255 bands generated by 21 ISSR primers, 99.61% were polymorphic and 99.71% of 344 bands amplified by 30 SRAP primers were polymorphic. The tested high genetic diversities show that the average Nei’s genetic diversity (H) were 0.1519 and 0.1624, and average Shannon’s information index (I) were 0.2248 and 0.2400 in ISSR and SRAP analyses, respectively. Unweighted pair group method with arithmetic mean (UPGMA) dendrograms of the nine populations were divided into two main groups. The ISSR and SRAP analyses values of gene differentiation (G _ST, 0.5955, 0.5486, respectively) indicate that high variation exists among the nine populations, likely due to external interferences and limitation of gene flow (N _m?=?0.3397, 0.4114). Our study indicated that human activities and herbivore overgrazing had influenced the natural Hizikia populations and that the understanding of population genetics would be helpful in sustainable utilization and biomass conservation of Sargassaceae resources.     

蛇神经毒素的研究进展

蛇毒是由许多种蛋白质、多肽、酶类以及其他小分子物质组成的混合物.在蛇毒中已经分离了许多种毒素分子,其中有一大类分子对哺乳动物的神经系统具有毒性效应,习惯上把这类分子成为蛇神经毒素.蛇神经毒素根据其作用位点的不同可以分为四大类:突触前蛇神经毒素、突触后蛇神经毒素、抗胆碱酯酶的蛇神经毒素和离子通道蛇神经毒素.许多蛇神经毒素已经分离纯化并进行了结构与功能的研究,几十近百种蛇神经毒素一级结构和空间结构已经得到测定.近几年来一些蛇神经毒素的基因文库以及cDNA文库已经构建出来,从中分离出的基因已经用于重组蛇神经毒素的生产研究.蛇神经毒素的分子结构与其功能具有较好的对应关系,即作用机制相同的毒素具有类似的空间结构.天然的蛇神经毒素以及重组的蛇神经毒素都已广泛应用于理论研究和一些临床应用.分离新的蛇神经毒素及其基因以及根据需要设计新的蛇神经毒素分子已成为该领域的热点,采用生物工程的方法规模生产蛇神经毒素也是当前及今后的研究方向.     

中国新记录属--大蓟马属分类研究(缨翅目:管蓟马科)

报道中国新记录属大蓟马属Megathrips Targioni—Tozzetti及新记录种宽腹大蓟马Megathrips lativentris（Heeger,1852）,并记述1新种——黑角大蓟马Megathrips antennatus．sp．nov．。     

含有母性影响遗传基因的连锁定位系的构建

母性影响遗传基因由于其杂交后代的表型受母本基因型的影响, 而不能直接反映当代个体的基因型, 这给连锁定位测交亲本(三隐性或双隐性系统)的培育带来困难, 从而影响这类基因的定位研究。设计了一套杂交培育方案, 其核心是使母性影响遗传基因先纯合, 再使非母性影响遗传基因纯合。采用所设计的方案, 成功培育了家蚕第13连锁群的赤蚁(ch)、无鳞毛翅(nlw, 新突变)和褐色卵t (b-t, 母性影响遗传)的三隐性系统, 并培育了第19连锁群的狭胸(nb)和第二肾形卵(ki-2, 母性影响遗传, 待定位突变基因)的双隐性系统。