GENPLAT: an Automated Platform for Biomass Enzyme Discovery and Cocktail Optimization

The high cost of enzymes for biomass deconstruction is a major impediment to the economic conversion of lignocellulosic feedstocks to liquid transportation fuels such as ethanol. We have developed an integrated high throughput platform, called GENPLAT, for the discovery and development of novel enzymes and enzyme cocktails for the release of sugars from diverse pretreatment/biomass combinations. GENPLAT comprises four elements: individual pure enzymes, statistical design of experiments, robotic pipeting of biomass slurries and enzymes, and automated colorimeteric determination of released Glc and Xyl. Individual enzymes are produced by expression in Pichia pastoris or Trichoderma reesei, or by chromatographic purification from commercial cocktails or from extracts of novel microorganisms. Simplex lattice (fractional factorial) mixture models are designed using commercial Design of Experiment statistical software. Enzyme mixtures of high complexity are constructed using robotic pipeting into a 96-well format. The measurement of released Glc and Xyl is automated using enzyme-linked colorimetric assays. Optimized enzyme mixtures containing as many as 16 components have been tested on a variety of feedstock and pretreatment combinations.GENPLAT is adaptable to mixtures of pure enzymes, mixtures of commercial products (e.g., Accellerase 1000 and Novozyme 188), extracts of novel microbes, or combinations thereof. To make and test mixtures of ˜10 pure enzymes requires less than 100 μg of each protein and fewer than 100 total reactions, when operated at a final total loading of 15 mg protein/g glucan. We use enzymes from several sources. Enzymes can be purified from natural sources such as fungal cultures (e.g., Aspergillus niger, Cochliobolus carbonum, and Galerina marginata), or they can be made by expression of the encoding genes (obtained from the increasing number of microbial genome sequences) in hosts such as E. coli, Pichia pastoris, or a filamentous fungus such as T. reesei. Proteins can also be purified from commercial enzyme cocktails (e.g., Multifect Xylanase, Novozyme 188). An increasing number of pure enzymes, including glycosyl hydrolases, cell wall-active esterases, proteases, and lyases, are available from commercial sources, e.g., Megazyme, Inc. (www.megazyme.com), NZYTech (www.nzytech.com), and PROZOMIX (www.prozomix.com).Design-Expert software (Stat-Ease, Inc.) is used to create simplex-lattice designs and to analyze responses (in this case, Glc and Xyl release). Mixtures contain 4-20 components, which can vary in proportion between 0 and 100%. Assay points typically include the extreme vertices with a sufficient number of intervening points to generate a valid model. In the terminology of experimental design, most of our studies are "mixture" experiments, meaning that the sum of all components adds to a total fixed protein loading (expressed as mg/g glucan). The number of mixtures in the simplex-lattice depends on both the number of components in the mixture and the degree of polynomial (quadratic or cubic). For example, a 6-component experiment will entail 63 separate reactions with an augmented special cubic model, which can detect three-way interactions, whereas only 23 individual reactions are necessary with an augmented quadratic model. For mixtures containing more than eight components, a quadratic experimental design is more practical, and in our experience such models are usually statistically valid.All enzyme loadings are expressed as a percentage of the final total loading (which for our experiments is typically 15 mg protein/g glucan). For "core" enzymes, the lower percentage limit is set to 5%. This limit was derived from our experience in which yields of Glc and/or Xyl were very low if any core enzyme was present at 0%. Poor models result from too many samples showing very low Glc or Xyl yields. Setting a lower limit in turn determines an upper limit. That is, for a six-component experiment, if the lower limit for each single component is set to 5%, then the upper limit of each single component will be 75%. The lower limits of all other enzymes considered as "accessory" are set to 0%. "Core" and "accessory" are somewhat arbitrary designations and will differ depending on the substrate, but in our studies the core enzymes for release of Glc from corn stover comprise the following enzymes from T. reesei: CBH1 (also known as Cel7A), CBH2 (Cel6A), EG1(Cel7B), BG (β-glucosidase), EX3 (endo-β1,4-xylanase, GH10), and BX (β-xylosidase).     

固定化纤维素酶对预处理纤维素原料水解效果的影响

以壳聚糖为载体用交联法制备固定化纤维素酶,考察固定化纤维素酶对蒸爆、球磨、超声波、喷淋、高温预处理玉米秸秆纤维素原料的酶解效果.结果表明:物料经蒸爆预处理后酶水解效率最高可以达到95%,球磨预处理水解效率次之,达到60%.用电镜和FT-IR对处理前后秸秆结构进行表征分析,证明预处理对物料的物理结构及化学组成有一定的影响.蒸爆法和球磨法可以使物料致密的天然结构彻底破坏,从而增加物料的比表面积;蒸爆预处理可以使纤维素内部氢键和官能团改变,使物料更易于酶解.     

黑曲霉固态发酵及酶解玉米皮

以玉米提取淀粉后的玉米皮渣为主要原料,采用黑曲霉固态发酵法产酶再酶解的二步法降解玉米皮中纤维素类物质。经Plackett-Burman法及响应面设计优化发酵条件得:温度30℃,接种量10%,初始水分体积分数60%,物料厚度2.47 cm,初始pH 5.79,发酵时间6 d;滤纸比酶活可达11.01 U/g,较原始酶活提高了40.61%;产酶结束后加入pH 4.8醋酸-醋酸钠缓冲液,置于50℃下酶解144 h,中性洗涤纤维与酸性洗涤纤维降解率分别为46.09%、48.82%,还原糖质量分数达到9.02%。     

Double chitin synthetase mutants from the corn smut fungus Ustilago maydis

Using genetic crosses between single chs mutants of Ustilago maydis inoculated into maize ( Zea mays ) seedlings, two classes of double mutants affected in genes coding for chitin synthetases were isolated: chs3 / chs4 , and chs4 / chs5 . Analysis of the mutants showed almost no change in their phenotype compared with wild-type strains. Growth rate, effect of stress conditions, dimorphic transition and mating were not affected. The only salient differences were increased sensitivity to osmotics at acid pH, and decrease in chitin synthetase activity, especially when measured with CO²⁺, and in chitin content. Most significant was a decrease in virulence, although this appeared to be due a factor unrelated to CHS genes. These data can be taken as further evidence that multigenic control of chitin synthetase in fungi operates as a safety mechanism to guarantee fungal viability in changing and hostile environmental conditions.     

Spatio-temporal dynamics of corn rootworm, Diabrotica spp., adults and their spatial association with environment

Multiyear and multilocation studies were conducted to investigate the within‐ and the between‐year spatial dynamics of corn rootworms Diabrotica spp. (Coleoptera: Chrysomelidae), adults and their spatial associations with environmental factors in cornfields. Grid‐based spatial sampling was conducted by trapping adults emerging from the soil (i.e., ‘emergence‐trap counts’) and by counting the number of adults in the ear zone of corn plant (i.e., ‘ear‐zone count’). Spatial analysis with distance indices (SADIE) was used to determine spatial distribution patterns and to investigate spatial associations. Ear‐zone counts and emergence‐trap counts were spatially dynamic within a year and more frequently aggregated in the middle of growing seasons and random early and late in the season. However, during the peak population periods, spatial distribution of ear‐zone counts and emergence‐trap counts were significantly consistent between years, indicating predictability of future spatial distributions. Spatial distribution of emergence‐trap counts and ear‐zone counts were positively associated with soil moisture and presence of corn plants with silks, respectively. This study demonstrated that within‐year spatial distribution patterns were dynamic and that there was between‐year spatial consistency of corn rootworm adult distributions. Such information can be used to improve preventative management of corn rootworms.     

Fumonisin production by <Emphasis Type="Italic">Fusarium</Emphasis> species isolated from freshly harvested corn in Iran

Fifty-one strains of Fusarium verticillioides and F. proliferatum isolated from corn collected from four different geographic areas in Iran, namely Fars, Khuzestan, Kermanshah and Mazandaran (an endemic oesophageal cancer (OC) area) were evaluated for their ability to produce fumonisins B₁ (FB₁), B₂ (FB₂) and B₃ (FB₃) in corn culture. Fumonisin levels were determined by high-performance liquid chromatography. All tested strains of F. verticillioides and F. proliferatumproduced fumonisins within a wide range of concentrations, 197–9661 g/g, 18–1974 g/g, and 21–1725 g/g for FB₁, FB₂, and FB₃, respectively. The highest mean concentrations of FB₁, FB₂, and FB₃ were 3897, 806 and 827 g/g, respectively. Overall, 61% of the F. verticillioides and F. proliferatum strains produced higher levels of FB₃ than FB₂. The mean ratios of FB₁:FB₂, FB₁:FB₃ and FB₁:total fumonisins were 8, 7 and 0.7 for F. verticillioides and 5.7, 10.7 and 0.7 for F. proliferatum, respectively. Significant differences in some of the meteorological data (rainfall, relative humidity and minimum temperature) from the four provinces were observed. Fumonisin levels produced by F. verticillioides strains isolated from Khuzestan province (tropical zone) were significantly (P < 0.01) higher than the other three provinces. This is the first report of the fumonisin-producing ability of F.verticillioides and F. proliferatum strains isolated from corn harvested from different geographic areas in Iran.     

Tolerance of Bt corn (MON 810) to maize stem borer, Chilo partellus (Lepidoptera: Pyralidae)

Transgenic corn (MON 810), expressing the Bacillus thuringiensis (Bt) protein, Cry1Ab, was evaluated under greenhouse conditions for its tolerance to the maize stem borer, Chilo partellus. Bt corn (MON 810) provided effective protection against the stem borer even under a high level of larval infestation in the greenhouse. The observed tolerance is examined and discussed in the light of the susceptibility of C. partellus to the Cry1Ab protein in laboratory bioassays. The implications of the tissue concentrations of Cry1Ab in MON 810, and baseline susceptibility recorded in the current study, for insect-resistance management are discussed.     

Chemical composition and rumen degradability of three corn hybrids treated with insecticides against the European corn borer (Ostrinia nubilalis)

This experiment determined the chemical composition, rumen degradability (aNDF in stalks and starch in kernels) and in vitro gas production of kernels from three corn hybrids treated (TT) or not treated (control, CTR) with insecticides against the European corn borer (ECB, Ostrinia nubilalis). Two whole-plant silage hybrids belonging to the FAO rating 600 and 700 maturity class (S600 and S700, respectively) and one selected for grain production (G600, FAO rating 600, Dekalb-Monsanto Agricoltura S.p.A., Lodi, Italy) were sown in two main plots (TT and CTR) of an experimental field. Two subsequent treatments of pyrethroids (25 and 1.2 g/ha of cyfluthrin and deltamethrin, respectively) were applied to the TT plots. The insecticide treatment reduced the number of damaged plants (4.5 broken plants/plot versus 0.3 broken plants/plot, P<0.01) and increased the total grain yield by 11% (13.8 t/ha versus 12.4 t/ha), while hybrids did not differ. ECB larvae which bored into the stalk tunnels modified the chemical composition of stalks and kernels. In stalks, total sugars content (i.e. glucose, fructose, sucrose) was about twice that in TT versus CTR plants (123 g/kg versus 60 g/kg DM, P<0.01), while aNDF content was higher in CTR stalks (765 versus 702 g/kg DM, P<0.01). DM degradability after 48 h of incubation of stalks was higher in TT than in CTR, both in vitro (0.360 versus 0.298, P<0.01) and in situ (0.370 versus 0.298, P<0.05), while there were no differences in aNDF degradability. Kernels from TT plots contained less DM (615 g/kg versus 651 g/kg, P<0.01) and more CP (84 g/kg and 78 g/kg DM, P<0.05) than those from CTR plots, while in situ rumen starch disappearance and in vitro gas production were similar. Corn hybrid selected for yield of grain (G600) differed from S600 and S700 due to a higher (P<0.01) content of aNDF, ADF and lignin(sa) in the stalks, and a higher starch content (696 g/kg versus 674 and 671 g/kg DM, P<0.01) and CP (87 g/kg versus 77 and 76 g/kg DM, P<0.05) in grain. The G600 hybrid produced stalks with a lower (P<0.01) aNDF rumen degradability than the S600 and S700.     

玉米黄尾球跳甲的发生与防治

调查研究玉米黄尾球跳甲Sphaeroderma apicaleBaly的自然种群动态、自然种群生命表并对其药剂防治策略进行探讨。结果表明:玉米黄尾球跳甲在湖北宜昌1年发生1~2代,以成虫和蛹越冬。越冬代成虫4月中旬始现于田边的杂草,5月上旬迁入大田。百株成虫高峰出现在6月15日和8月7日;性比为0.491;卵始现于5月19日,产卵高峰出现在6月18日;平均卵量123.5粒,单雌最大卵量212粒;6月22日出现最高百株幼虫量。自然种群增长指数(I)为16.20。在发生盛期、高峰期、盛末期,玉米黄尾球跳甲被异角短胸姬小蜂Hemiptarsenus variconisGirault的寄生率分别为14.6,33.6和77.0%。马.辛硫磷1 000倍液和毒死蜱1 000倍液是防治玉米黄尾球跳甲比较理想的农药,校正防治效果分别为76.3%和66.5%;卵和1、2龄幼虫对化学药剂敏感性高,3龄幼虫对化学药剂敏感性低。结合以上的研究,提出玉米黄尾球跳甲的防治策略。     

转Bt基因抗虫玉米对亚洲玉米螟幼虫几种主要酶系活性的影响

研究了表达Cry1Ab杀虫蛋白的转Bt基因抗虫玉米对亚洲玉米螟Ostrinia furnacalis (uenée) 幼虫解毒酶、保护酶和中肠蛋白酶活性的影响,测定比较了取食转Bt基因玉米后幼虫体内α-乙酸萘酯酶、乙酰胆碱酯酶、谷胱甘肽S-转移酶、过氧化氢酶、超氧化物歧化酶、中肠总蛋白酶、类胰蛋白酶和类胰凝乳蛋白酶的活力。结果表明,取食转Bt基因玉米48 h后亚洲玉米螟幼虫体内的α-乙酸萘酯酶、谷胱甘肽S转移酶活力明显低于对照;而乙酰胆碱酯酶活力显著高于对照,在取食48 h、60 h和72 h的活力分别是对照的2.00、1.50和2.50倍。保护酶系、中肠总蛋白酶、弱碱性类胰蛋白酶和类胰凝乳蛋白酶的活性在取食48 h后明显受到抑制;但强碱性类胰蛋白酶的活性显著高于对照,取食48 h、60 h和72 h的活力分别是对照的4.00、1.67和1.33倍。乙酰胆碱酯酶和强碱性类胰蛋白酶可能与亚洲玉米螟对Bt的抗性有关。