Genetic Engineering of Energy Crops: A Strategy for Biofuel Production in China

Biomass utilization is increasingly considered as a practical way for sustainable energy supply and long-term environment care around the world.In concerns with food security in China,starch or sugar-based bioethanol and edible-oil-derived biodiesel are harshly restricted for large scale production.However,conversion of lignocellulosic residues from food crops is a potential alternative.Because of its recalcitrance,current biomass process is unacceptably expensive,but genetic breeding of energy crops is a p...     

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 Bioen-     

Genetic Engineering of Algae for Enhanced Biofuel Production

There are currently intensive global research efforts aimed at increasing and modifying the accumulation of lipids, alcohols, hydrocarbons, polysaccharides, and other energy storage compounds in photosynthetic organisms, yeast, and bacteria through genetic engineering. Many improvements have been realized, including increased lipid and carbohydrate production, improved H₂ yields, and the diversion of central metabolic intermediates into fungible biofuels. Photosynthetic microorganisms are attracting considerable interest within these efforts due to their relatively high photosynthetic conversion efficiencies, diverse metabolic capabilities, superior growth rates, and ability to store or secrete energy-rich hydrocarbons. Relative to cyanobacteria, eukaryotic microalgae possess several unique metabolic attributes of relevance to biofuel production, including the accumulation of significant quantities of triacylglycerol; the synthesis of storage starch (amylopectin and amylose), which is similar to that found in higher plants; and the ability to efficiently couple photosynthetic electron transport to H₂ production. Although the application of genetic engineering to improve energy production phenotypes in eukaryotic microalgae is in its infancy, significant advances in the development of genetic manipulation tools have recently been achieved with microalgal model systems and are being used to manipulate central carbon metabolism in these organisms. It is likely that many of these advances can be extended to industrially relevant organisms. This review is focused on potential avenues of genetic engineering that may be undertaken in order to improve microalgae as a biofuel platform for the production of biohydrogen, starch-derived alcohols, diesel fuel surrogates, and/or alkanes.Interest in a variety of renewable biofuels has been rejuvenated due to the instability of petroleum fuel costs, the reality of peak oil in the near future, a reliance on unstable foreign petroleum resources, and the dangers of increasing atmospheric CO₂ levels. Photosynthetic algae, both microalgae and macroalgae (i.e., seaweeds), have been of considerable interest as a possible biofuel resource for decades (165). Several species have biomass production rates that can surpass those of terrestrial plants (41), and many eukaryotic microalgae have the ability to store significant amounts of energy-rich compounds, such as triacylglycerol (TAG) and starch, that can be utilized for the production of several distinct biofuels, including biodiesel and ethanol. It is believed that a large portion of crude oil is of microalgal origin, with diatoms being especially likely candidates, considering their lipid profiles and productivity (153). If ancient algae are responsible for creating substantial crude oil deposits, it is clear that investigation of the potential of living microalgae to produce biofuels should be a priority. Microalgae are especially attractive as a source of fuel from an environmental standpoint because they consume carbon dioxide and can be grown on marginal land, using waste or salt water (41). In addition, it may be possible to leverage the metabolic pathways of microalgae to produce a wide variety of biofuels (Fig. 1). In contrast to corn-based ethanol or soy/palm-based biodiesel, biofuels derived from microalgal feedstocks will not directly compete with the resources necessary for agricultural food production if inorganic constituents can be recycled and saltwater-based cultivation systems are developed.Open in a separate windowFig. 1.Microalgal metabolic pathways that can be leveraged for biofuel production. ER, endoplasmic reticulum.However, several technical barriers need to be overcome before microalgae can be used as an economically viable biofuel feedstock (139). These include developing low-energy methods to harvest microalgal cells, difficulties in consistently producing biomass at a large scale in highly variable outdoor conditions, the presence of invasive species in large-scale ponds, low light penetrance in dense microalgal cultures, the lack of cost-effective bioenergy carrier extraction techniques, and the potentially poor cold flow properties of most microalga-derived biodiesel. To advance the utilization of microalgae in biofuel production, it is important to engineer solutions to optimize the productivity of any microalgal cultivation system and undertake bioprospecting efforts to identify strains with as many desirable biofuel traits as possible. Over 40,000 species of algae have been described, and this is likely only a small fraction of the total number of available species (75). The U.S. Department of Energy''s Aquatic Species Program analyzed approximately 3,000 different microalgae for their potential to produce biofuels, and numerous additional species have subsequently been investigated (165). Although these efforts demonstrated that many species of microalgae have properties that are desirable for biofuel production, most have drawbacks that have prevented the emergence of an economically viable algal biofuel industry. It is postulated that a light-harvesting footprint of at least 20,000 square miles will be required to satisfy most of the current U.S. transportation fuel demand (41). Therefore, even modest improvements in photon conversion efficiencies will dramatically reduce the land area and cost required to produce biofuels. Consequently, continued bioprospecting efforts and the development and engineering of select microalgal strains are required to improve the yields of bioenergy carriers. Current commercial agriculture crops have been cultivated for thousands of years, with desired traits selected over time. It stands to reason that with microalgae, it would be beneficial to use genetic engineering in an attempt to bypass such a lengthy selection process. However, despite the recent advances in biotechnological approaches, the full potential of genetic engineering in some microalgal species, particularly diploid diatoms, can be fully realized only if conventional breeding methods become firmly established, thereby allowing useful traits or mutations to be easily combined (5, 24, 25). Since the topic of microalgal sexual breeding is beyond the scope of this review, we will instead focus on genetic engineering approaches that could be utilized in the industry''s efforts to improve microalgae as a source of biofuels.     

作物抗病基因工程研究进展

控制植物病害的关键,取决于对植物与病原菌相互作用的分子机理的了解。将抗病基因、信号传导／调控基因、抗菌蛋白基因等导入拟改良的作物、选育抗病新品系,是当前作物抗病基因工程研究的主要策略。本文介绍利用植物抗病反应中系列重要基因进行作物抗病基因工程的研究进展,讨论了目前作物抗病基因工程中存在的问题及其解决的方法。     

农作物抗除草剂基因工程

概述抗除草剂作物基因工程抗性基因的来源,产生抗性的机理,抗除草剂外源基因的导入方法,并对目前普遍关注的抗除草剂作物的安全性问题进行了讨论,提出了抗除草剂作物研究及开发中应注意的问题。     

Photosynthetic Energy Conversion Efficiency: Setting a Baseline for Gauging Future Improvements in Important Food and Biofuel Crops

The conversion efficiency (ε_c) of absorbed radiation into biomass (MJ of dry matter per MJ of absorbed photosynthetically active radiation) is a component of yield potential that has been estimated at less than half the theoretical maximum. Various strategies have been proposed to improve ε_c, but a statistical analysis to establish baseline ε_c levels across different crop functional types is lacking. Data from 164 published ε_c studies conducted in relatively unstressed growth conditions were used to determine the means, greatest contributors to variation, and genetic trends in ε_c across important food and biofuel crop species. ε_c was greatest in biofuel crops (0.049–0.066), followed by C₄ food crops (0.046–0.049), C₃ nonlegumes (0.036–0.041), and finally C₃ legumes (0.028–0.035). Despite confining our analysis to relatively unstressed growth conditions, total incident solar radiation and average growing season temperature most often accounted for the largest portion of ε_c variability. Genetic improvements in ε_c, when present, were less than 0.7% per year, revealing the unrealized potential of improving ε_c as a promising contributing strategy to meet projected future agricultural demand.Substantial increases in yield are needed to feed and fuel the world’s growing human population. With an estimated population of nine billion people by the middle of this century (Lutz and Samir, 2010) and rising affluence resulting in greater consumption of grain-fed animal products (Cirera and Masset, 2010), different studies predict that, by midcentury, global crop production will need to increase 60% to 120% over 2005 levels without the expansion of agricultural land area (Tilman et al., 2011; Alexandratos and Bruinsma, 2012).Doubling yields in major food and fuel crops requires considerable effort, especially as yields are beginning to plateau in many major food crops. Yield increases necessary for doubling productivity by midcentury are estimated at 1.16% to 1.31% each year in all cereals (Hall and Richards, 2013), 1.7% per year in wheat (Triticum aestivum; Rosegrant and Agcaoili, 2010), and 2.4% (noncompounding average per year) across all major grain crops (Ray et al., 2013). However, global mean increases from the past 20 to 30 years suggest that yield gains in rice (Oryza sativa) and wheat are approximately 1% (Lopes et al., 2012; Manès et al, 2012; Ray et al., 2013) and declining in some areas of the world (Cassman et al., 2010; Fischer and Edmeades, 2010; Long and Ort, 2010; Ray et al., 2013). Global yearly increases are estimated at 1.3% in soybean (Glycine max) and 1.6% in maize (Zea mays), with similar concerns that yield trends may also be decreasing in some major growing regions (Lobell and Gourdji, 2012; Ray et al., 2013).Efforts to increase yields in the next few decades must also account for environmental and sustainability goals (Sayer et al., 2013) as well as heightened environmental stresses predicted to occur due to climate change, which are already responsible for some of the stagnation in yield increases. Anthropogenic sources of greenhouse gases have caused an approximately 1°C increase in land surface temperatures since 1900, and global mean surface temperatures are likely to increase by up to 2.4°C to 4.8°C by the end of the century (IPCC, 2013). Drought is also expected to become more frequent and intense in many regions of the world (Dai, 2011; IPCC, 2013). Of the variability present in major food crop yield gains, 30% can be explained by climate change alone (Lobell and Field, 2007), with drastic decreases in barley (Hordeum vulgare), maize, rice, sorghum (Sorghum bicolor), soybean, and wheat yields as average growing season temperatures surpass the temperature optimum for each crop (Lobell and Gourdji, 2012). Current levels of atmospheric CO₂ concentration [CO₂] are the highest they have been in at least 800,000 years (IPCC, 2013). Elevated [CO₂] increases water use efficiency (Ainsworth and Long, 2005, Bernacchi et al., 2007, Leakey et al., 2009), but probably not to an extent that would mitigate the resulting reductions in yield caused by higher temperature and higher vapor pressure deficit (Ort and Long, 2014). Additionally, any fertilization effects on C₃ yields due to elevated [CO₂] would be at least in part negated by drought and temperature stress, leaving yield increases far from optimal (Long et al., 2006a; Lobell and Gourdji, 2012).     

基因工程在能源植物改良中的应用

利用基因工程技术改良能源植物,对降低能源植物向生物燃料(生物乙醇、生物柴油)的转化成本、提高能源转化效率有着非常重要的意义。目前,基因工程技术已被广泛应用于提高植物总的生物产量、降低或改变植物木质素的含量与成分、在植物体中大量表达纤维素降解酶、提高油料植物的产油量以及改变植物油酯的组成成分等方面的研究。概述了利用基因工程技术在以上方面对能源植物进行改良已取得的进展,讨论了现存问题及未来的发展前景。     

Engineering an Isoprenoid Pathway in Escherichia coli for Production of 2-Methyl-3-buten-2-ol: A Potential Biofuel

2-Methyl-3-buten-2-ol (MBO) is a natural volatile 5-carbon alcohol produced by several pine species that have the potential to be used as biofuel. MBO has a high energy content making it superior to ethanol in terms of energy output, and due to its volatility and lower solubility in water, MBO is easier to recover than ethanol. Pine’s MBO synthase enzyme utilizes the intermediate dimethylallyl pyrophosphate (DMAPP) produced by the methyl-erythritol-4-phosphate isoprenoid pathway for the production of MBO. In this study, we performed metabolic engineering of Escherichia coli to express an alternate mevalonate dependent pathway for production of DMAPP, along with a codon optimized Pinus sabiniana MBO synthase gene. This heterologous expressed pathway carried out the conversion of an acetyl CoA precursor to DMAPP leading to production of MBO.     

Implications of Three Biofuel Crops for Beneficial Arthropods in Agricultural Landscapes

Production of biofuel feedstocks in agricultural landscapes will result in land use changes that may have major implications for arthropod-mediated ecosystem services such as pollination and pest suppression. By comparing the abundance and diversity of insect pollinators and generalist natural enemies in three model biofuel crops: corn, switchgrass, and mixed prairie, we tested the hypothesis that biofuel crops comprised of more diverse plant communities would support increased levels of beneficial insects. These three biofuel crops contained a diverse bee community comprised of 75 species. Overall, bees were three to four times more abundant in switchgrass and prairie than in corn, with members of the sweat bee (Halictidae) and small carpenter bee (Ceratina spp.) groups the most abundant. Switchgrass and prairie had significantly greater bee species richness than corn during the July sampling period. The natural enemy community at these sites was dominated by lady beetles (Coccinellidae), long-legged flies (Dolichopodidae), and hover flies (Syrphidae) which varied in their response to crop type. Coccinellids were generally most abundant in prairie and switchgrass, with the exception of the pollen feeding Coleomegilla maculata that was most abundant in corn. Several rare or declining coccinellid species were detected in prairie and switchgrass sites. Dolichopodidae were more abundant in prairie and switchgrass while Syrphidae showed no significant response to crop type. Our results indicate that beneficial insects generally responded positively to the increased vegetational diversity of prairie and switchgrass sites; however, when managed as a dedicated biofuel crop, plant and arthropod diversity in switchgrass may decrease. Our findings support the hypothesis that vegetationally diverse biofuel crops support higher abundance and diversity of beneficial insects. Future policy regarding the production of biofuel feedstocks should consider the ecosystem services that different biofuel crops may support in agricultural landscapes.     

Prospects for Cellulosic Biofuel Production in the Northeastern United States: A Scenario Analysis

Secure access to energy and food are two of the challenges facing the Northeast region of the United States. Traditional biofuel feedstocks, such as corn and oil seed, are able to satisfy energy requirements. However, they compete with food production for desirable land and water resources and, in any case, are not likely to exploit the region's current comparative advantages. This study investigates a potential solution to the energy security problem in the Northeast: biofuel from advanced feedstock in the form of net forest growth and woody wastes, of which the region has abundant endowments. The federal government has committed to requiring 79.5 billion liters (BL) of advanced biofuel production annually by 2022. We evaluate both the physical capacity for its production and its cost competitiveness using an input‐output model of consumption, production, and trade in the 13‐state region. The model minimizes resource use required to satisfy given consumer demand using alternative technological options and subject to resource constraints. We compile data from the technical literature quantifying state‐level biofuel feedstock endowments and the technological requirements for cellulosic ethanol production. We find that exploiting the region's endowment of cellulosic feedstock requires either making the price of biofuels competitive with gasoline through subsidies or restricting imports of gasoline. Based on this initial investigation, we conclude that the region can produce significant amounts of advanced biofuel, up to 20.28 BL of cellulosic ethanol per year, which could displace nearly 12.5% of the gasoline that is now devoted to motorized transport in the region.     

中国作物野生近缘植物资源

中国地域辽阔,气候多样,地形复杂,土壤类型繁多,加之农业历史悠久,不仅作物种类众多,而且很多作物都有其野生近缘种,从而使得中国成为世界作物的重要起源中心之一。本文在介绍中国作物种类的同时,列出了各类作物的野生近缘植物资源,为进一步深入研究和开发利用这些资源,以及国家制定野生植物保护有关政策提供基础数据和信息。     

Genetic Engineering with Bacillus thuringiensis and Conventional Approaches for Insect Resistance in Crops

The aim of resistance management is to slow and ideally reverse the development of resistance in the pest population. Since 1996, million of acres of crops have been planted that are genetically engineered with Bacillus thuringiensis (Bt) for insect resistance. The novelty for resistance management is that with Bt technology it is possible to control the principal force in an agroecosystem microevolutionary process from the outset, i.e., selection pressure. In Bt crops, the toxin can be expressed constitutively at a relatively constant dose or expression of the toxin can be restricted to specific crop stages, tissues, or both. Here we propose that more precise control of selection eases the practical application of resistance management strategies (high-dose–refugia) compared to other resistance strategies. The population genetics and ecological and operational factors related to the high-dose–refugia strategy currently used for Bt crops are also reviewed.     

作物品种间杂种优势遗传分析的新方法

本文提出了分析双列杂交试验资料的两个遗传模型。第一个模型包括加性、显性和母体效应;第二个模型只包括简单的加性和显性效应。还介绍了分析杂种优势、估算遗传方差分量以及预测遗传效应值的相应统计分析方法。用所介绍的遗传模型和分析方法以及常用的Griffing配合力分析方法,分析了棉花6个品种双列杂交的产量性状,并进一步比较了不同方法的分析结果。采用本文所介绍的遗传模型和分析方法,可以克服用Griffing的配合力模型及其方法分析杂种优势和配合力遗传表现所存在的局限性。     

表面展示工程在酒精发酵方面的应用

目前,表面展示工程作为一种新型的极具应用潜力的技术手段,在组合文库的筛选、蛋白质工程、燃料乙醇的生产和生物修复等技术中已经扮演了极其重要的角色.介绍了表面展示工程在酒精发酵方面的应用,重点阐述酒精发酵过程中酶类展示原理和全细胞催化的应用前景.     

Biological Nitrogen Fixation in Sugar Cane: A Key to Energetically Viable Biofuel Production

The advantages of producing biofuels to replace fossil energy sources are derived from the fact that the energy accumulated in the biomass is captured directly from photosynthesis and is thus renewable, and that the cycle of carbon dioxide fixation by the crop, followed by burning of the fuel makes no overall contribution to atmospheric CO₂ or, consequently, to global warming. However, these advantages are negated if large quantities of fossil fuels need to be used to grow or process the biofuel crop. In this regard, the Brazilian bioethanol program, based on the fermentation/distillation of sugar cane juice, is particularly favorable, not only because the crop is principally hand harvested, but also because of the low nitrogen fertilizer use on sugar cane in Brazil. Recent ¹⁵N and N balance studies have shown that in some Brazilian cane varieties, high yields are possible without N fertilization because the plants are able to obtain large contributions of nitrogen from plant-associated biological N₂ fixation (BNF). The N₂-fixing acid-tolerant bacterium Acetobacter diazotrophicus was first found to occur within roots, stems, and leaves of sugar cane. Subsequently, two species of Herbaspirillum also have been found to occur within the interior of all sugar cane tissues. The discovery of these, and other N₂-fixing bacteria that survive poorly in soil but thrive within plant tissue (endophytic bacteria), may account for the high BNF contributions observed in sugar cane. Further study of this system should allow the gradual elimination of N fertilizer use on sugar cane, at least in Brazil, and opens up the possibility of the extension of this efficient N₂-fixing system to cereal and other crops with consequent immense potential benefits to tropical agriculture.     

大肠杆菌产琥珀酸基因工程研究进展

生物合成琥珀酸摆脱了对不可再生战略资源石油的依赖,以其社会、经济和环境效益展现出良好的发展前景。野生型大肠杆菌的琥珀酸生产强度难以满足生物合成琥珀酸工业化的要求,但遗传背景清楚,容易改造。近年来,人们深入研究了大肠杆菌的琥珀酸代谢途径,通过强化大肠杆菌琥珀酸合成途径、抑制琥珀酸旁路代谢途径、构建产琥珀酸乙醛酸循环和有氧生产体系等多种基因工程策略,对大肠杆菌进行菌株改造和代谢进化筛选,提高了琥珀酸产量。综述了大肠杆菌产琥珀酸的基因工程研究进展。     

宏基因组技术在新型生物燃料生产酶制剂开发中的应用

随着石化燃料的日益减少,以植物生物质为原料的可再生生物燃料成为石化燃料的理想替代品。然而微生物降解生物质效率低下,是生物燃料生产过程中一大难题,因此开发效率高、稳定性强的微生物酶制剂显得尤为重要。近年来,宏基因组技术的发展为生物燃料的生产提供了多种新型酶制剂。宏基因组技术是直接提取环境样品中的总DNA,通过构建文库,筛选目的基因或功能基因的方法,在用于燃料生产的新型酶制剂的开发中发挥着重要作用。本文概述了宏基因组技术的实施策略,总结了包括纤维素酶、蛋白酶、酯酶、脂肪酶等多种酶资源开发的最新研究进展,并综合和讨论了通过酶法将木质纤维素等生物材料有效转化为生物燃料的途径,为新酶的开发提供了新思路。     

Genetic Engineering of Zymobacter palmae for Production of Ethanol from Xylose

Its metabolic characteristics suggest that Zymobacter palmae gen. nov., sp. nov. could serve as a useful new ethanol-fermenting bacterium, but its biotechnological exploitation will require certain genetic modifications. We therefore engineered Z. palmae so as to broaden the range of its fermentable sugar substrates to include the pentose sugar xylose. The Escherichia coli genes encoding the xylose catabolic enzymes xylose isomerase, xylulokinase, transaldolase, and transketolase were introduced into Z. palmae, where their expression was driven by the Zymomonas mobilis glyceraldehyde-3-phosphate dehydrogenase promoter. When cultured with 40 g/liter xylose, the recombinant Z. palmae strain was able to ferment 16.4 g/liter xylose within 5 days, producing 91% of the theoretical yield of ethanol with no accumulation of organic acids as metabolic by-products. Notably, xylose acclimation enhanced both the expression of xylose catabolic enzymes and the rate of xylose uptake into recombinant Z. palmae, which enabled the acclimated organism to completely and simultaneously ferment a mixture of 40 g/liter glucose and 40 g/liter xylose within 8 h, producing 95% of the theoretical yield of ethanol. Thus, efficient fermentation of a mixture of glucose and xylose to ethanol can be accomplished by using Z. palmae expressing E. coli xylose catabolic enzymes.