粗糙脉孢菌纤维素酶液体发酵优良形态突变体筛选

丝状真菌被广泛地用于包括纤维素酶在内的工业酶生产过程。在液体深层发酵中,丝状真菌菌丝形态直接影响发酵液的流变特性,进而与目标酶蛋白产量存在着重要的关联。目前,针对丝状真菌工业酶液体发酵菌丝形态的研究依然是从传统的发酵工程学角度出发,对与发酵水平紧密相关的形态、粘度等性状相关基因的认识远远不够。为了挖掘深层发酵中对丝状真菌发酵产酶性能具有重要影响的形态发育相关基因,以粗糙脉孢菌Neurospora crassa单基因突变体库中的95株形态突变株为研究对象,在结晶纤维素为碳源的条件下进行筛选,探寻与野生型菌株蛋白产量有显著差异的突变株。同时,对这些突变株的内切-β-1,4-葡聚糖酶酶活、β-葡萄糖苷酶酶活、发酵液粘度和菌丝干重进行了测定,并观察了发酵液中突变株的菌丝形态。实验结果表明,与野生型菌株相比,突变株SZY32、SZY35、SZY39和SZY43发酵液中蛋白浓度显著降低,突变株SZY11、SZY63、SZY69和SZY87发酵液中蛋白浓度显著性提高。值得注意的是,突变株SZY11和SZY43发酵液菌丝体主要形态为菌球状,其发酵液粘度分别降低75%和50%,突变株SZY87在发酵液中呈长丝状,发酵液粘度显著升高至少2倍。这些与产酶水平相关的形态、粘度基因的获得将有助于丝状真菌纤维素酶等工业酶高产工程菌株的理性构建。     

Cellulosic ethanol production: Progress,challenges and strategies for solutions

Lignocellulosic biomass is a sustainable feedstock for fuel ethanol production, but it is characterized by low mass and energy densities, and distributed production with relatively small scales is more suitable for cellulosic ethanol, which can better balance cost for the feedstock logistics. Lignocellulosic biomass is recalcitrant to degradation, and pretreatment is needed, but more efficient pretreatment technologies should be developed based on an in-depth understanding of its biosynthesis and regulation for engineering plant cell walls with less recalcitrance. Simultaneous saccharification and co-fermentation has been developed for cellulosic ethanol production, but the concept has been mistakenly defined, since the saccharification and co-fermentation are by no means simultaneous. Lignin is unreactive, which not only occupies reactor spaces during the enzymatic hydrolysis of the cellulose component and ethanol fermentation thereafter, but also requires extra mixing, making high solid loading difficult for lignocellulosic biomass and ethanol titers substantially compromised, which consequently increases energy consumption for ethanol distillation and stillage discharge, presenting another challenge for cellulosic ethanol production. Pentose sugars released from the hydrolysis of hemicelluloses are not fermentable with Saccharomyces cerevisiae used for ethanol production from sugar- and starch-based feedstocks, and engineering the brewing yeast and other ethanologenic species such as Zymomonas mobilis with pentose metabolism has been performed within the past decades. However strategies for the simultaneous co-fermentation of pentose and hexose sugars that have been pursued overwhelmingly for strain development might be modified for robust ethanol production. Finally, unit integration and system optimization are needed to maximize economic and environmental benefits for cellulosic ethanol production. In this article, we critically reviewed updated progress, and highlighted challenges and strategies for solutions.     

High‐level hemicellulosic arabinose predominately affects lignocellulose crystallinity for genetically enhancing both plant lodging resistance and biomass enzymatic digestibility in rice mutants

Rice is a major food crop with enormous biomass residue for biofuels. As plant cell wall recalcitrance basically decides a costly biomass process, genetic modification of plant cell walls has been regarded as a promising solution. However, due to structural complexity and functional diversity of plant cell walls, it becomes essential to identify the key factors of cell wall modifications that could not much alter plant growth, but cause an enhancement in biomass enzymatic digestibility. To address this issue, we performed systems biology analyses of a total of 36 distinct cell wall mutants of rice. As a result, cellulose crystallinity (CrI) was examined to be the key factor that negatively determines either the biomass enzymatic saccharification upon various chemical pretreatments or the plant lodging resistance, an integrated agronomic trait in plant growth and grain production. Notably, hemicellulosic arabinose (Ara) was detected to be the major factor that negatively affects cellulose CrI probably through its interlinking with β‐1,4‐glucans. In addition, lignin and G monomer also exhibited the positive impact on biomass digestion and lodging resistance. Further characterization of two elite mutants, Osfc17 and Osfc30, showing normal plant growth and high biomass enzymatic digestion in situ and in vitro, revealed the multiple GH9B candidate genes for reducing cellulose CrI and XAT genes for increasing hemicellulosic Ara level. Hence, the results have suggested the potential cell wall modifications for enhancing both biomass enzymatic digestibility and plant lodging resistance by synchronically overexpressing GH9B and XAT genes in rice.     

¹H-NMR-based metabonomic analysis of metabolic profiling in diabetic nephropathy rats induced by streptozotocin

Elucidation of the metabolic profiling in diabetic nephropathy (DN) rats is of great assistance for understanding the pathogenesis of DN. In this study, 1H-nuclear magnetic resonance (NMR)-based metabonomics combined with HPLC measurements was used to quantitatively analyze the metabolic changes in urine and kidney extracts from diabetic 2-wk and 8-wk rats induced by streptozotocin (STZ). Pattern recognition analysis of either urine or kidney extracts indicated that the two diabetic groups were separated obviously from the control group, suggesting that the metabolic profiles of the diabetic groups were markedly different from the control. The diabetic 8-wk rats showed lower levels of creatine, dimethylamine, and higher levels of ascorbate, succinate, lactate, citrate, allantoin, 2-ketoglutarate, and 3-hydrobutyrate (3-HB) in the urine samples. Moreover, the diabetic 8-wk rats displayed lower levels of succinate, creatine, myo-inositol, alanine, lactate, and ATP, and higher levels of 3-HB and glucose in the kidney extracts. The observed metabolic changes imply the enhanced pathways of either lipid or ketone body synthesis and decreased pathways of either tricarboxylic acid cycle or glycolysis in DN rats compared with the control. Our results suggest that the energy metabolic changes are associated with the pathogenic process of DN.     

Energy crop and biotechnology for biofuel production

Selection of energy crops is the first priority for large-scale biofuel production in China.As a major topic, it was extensively discussed in the Second International Symposium on Bioenergy and Biotechnology, held from October 16-19(th), 2010 in Huazhong Agricultural University(HZAU), Wuhan, China, with more than one hundred registered participants(Figure 1).     

水稻原生质体培养及植株再生的研究

由粳稻77-170品系及籼稻品种IR-50的细胞悬浮培养物游离的原生质体,用琼脂糖包埋于RY-2培养基中,发生了持续分裂。前者植板率达2.5％以上,二者最后都再生出植株。对游离和培养方法做了如下改进:1)采用两步法,即先用果胶酶,再用果胶酶和纤维素酶的混合酶进行游离,可避免原生质体发生融合并获得高质量的原生质体;2)悬浮细胞培养基中加入ABA有利于原生质体的存活和分裂;3)琼脂糖包埋培养可大大提高植板率;4)用较高渗透压的培养基培养原生质体再生的细胞团及愈伤组织,可提高植株再生频率。由于这两个品种(系)的培养物都已继代一年半之久,再生植株均为白化苗。这是迄今第一个由籼稻原生质体再生植株的报道。     

籼稻基因枪转化的研究

对国内外１７个籼稻品种进行了基因枪转化,研究了有利于籼稻愈伤组织诱导和生长的培养条件和筛选程序,１５个品种获得了潮霉素抗性愈伤组织,５个品种再生了植株,包括当前国际上推广的ＩＲ系统及国内的优良品种和恢复系,分子生物学证据证明潮霉素基因已经整合入籼稻的基因组。最高植株转化频率接近一般粳稻水平,多数低于粳稻水平。这为建立籼稻的转化系统打下了基础。     

拟南芥纤维素合酶基因时空表达模式与功能预测

纤维素是细胞壁的主要组成成分, 研究纤维素合成可以从源头上解决关于高效降解纤维素的问题。该研究通过综合拟南芥(Arabidopsis thaliana)纤维素合酶基因(AtCESA)家族的进化和芯片表达分析及根据拟南芥全生育期GUS染色结果分析纤维素合酶基因的时空表达模式, 发现拟南芥纤维素合酶基因AtCESA1, 3, 6以及AtCESA4, 7, 8分别参与细胞壁初生壁和次生壁的合成并存在明显的共表达现象。其中, AtCESA1, 3, 6在全生育期表达, AtCESA4, 7, 8主要在根、茎和叶脉等次生壁细胞中表达。AtCESA5和AtCESA6、AtCESA2和AtCESA9以及AtCESA1和AtCESA10等基因对均有基因重复作用。根据AtCESA家族基因表达模式和分子演化关系可以推测, AtCESA5对AtCESA6以及AtCESA9对AtCESA2可能分别存在功能冗余。此外, AtCESA9的表达具明显的组织特异性。上述研究结果为深入认识拟南芥纤维素合酶基因的功能奠定了基础。     

Time-Dependent Lactate Production and Amino Acid Utilization in Cultured Astrocytes Under High Glucose Exposure

Accumulating investigations have focused on the severity of central nervous system (CNS) complications in diabetic patients. The effects of the high glucose (HG) probably attribute to the metabolic disturbances in CNS. Astrocytes, with powerful ability of metabolic regulation, play crucial roles in physiological and pathological processes in CNS. Hence, an in-depth analysis as to metabolic alterations of astrocytes exposure to HG would facilitate to explore the underlying pathogenesis. In this study, the ¹H NMR-based metabonomic approach was performed to characterize the metabolic variations of intracellular metabolites and corresponding culture media in a time-dependent manner. Our results revealed a significant elevation in lactate production and release. Four amino acids, leucine, isoleucine, methionine and tyrosine, were the most important metabolites for utilization. Also, profound disturbances of several metabolic pathways, including osmoregulation, energy metabolism, and cellular biosynthesis were observed. In that sense, the detailed information of astrocyte metabolism under HG exposure provides us a comprehensive understanding of the intrinsic metabolic disorders in CNS during hyperglycemia or diabetes.     

锈毛草莓积雪草合剂对STZ小鼠降血糖作用研究

采用75%乙醇萃取锈毛草莓及积雪草,制备成合剂,通过STZ诱导建立糖尿病小鼠模型,随机分为模型组、低剂量组及高剂量组,分别以蒸馏水、4及8 g·kg~(-1)锈毛草莓积雪草合剂灌胃,1次·d~(-1),持续4周,另设正常组作为对照。探究绣毛草莓积雪草合剂对糖尿病小鼠的降血糖效果及作用机制。结果表明:(1)不同剂量的锈毛草莓积雪草合剂均能显著降低糖尿小鼠血糖值,提高糖耐量并减轻其多食、多饮、多尿的症状。(2)低剂量组小鼠血清TC、TG含量较模型组有显著提高(P0.01或P0.05),肝脏SOD、CAT活性显著提高(P0.05),MDA含量明显降低。(3)低剂量黄毛草莓积雪草合剂效果优于二甲双胍,毒性实验显示锈毛草莓积雪草合剂实属无毒。该研究结果表明锈毛草莓积雪草合剂具有降血糖作用,同时能够降低糖尿病小鼠的血脂水平,其作用机制可能与提高肝脏抗氧化能力有关。