体外生物转化合成酶途径生产生物燃料翻译.doc

Biofuel production by in vitro synthetic enzymatic pathway biotransformation体外生物转化合成酶途径生产生物燃料Cell-free synthetic pathway biotransformation (SyPaB) is the implementation of complicated biochemical reactions by in vitro assembling a number of enzymes or their complexes and coenzymes. 无细胞生物转化合成途径（SyPaB）是复杂的生化反应过程。它在体外装配一系列的酶、酶的复合物和辅酶。Assembly of numerous enzymes without cellular membrane, gene regulation, or undesired pathway can circumvent some of the obstacles to modifying living microorganisms. 无细胞膜，基因调控，或未知的过程的酶的装配过程会规避一些障碍修正活的微生物。Several synthetic pathways for the production of liquid biofuels alcohols and hydrocarbon precursors (polyols) as well as gaseous biofuel hydrogen have been presented. 有人已经报道了几种用于生产生物燃料的合成，如液体生物燃料： 醇类和烃类前体（多元醇）以及气态燃料 ： 氢气。The present constraints to SyPaB include the lack of stable enzymes as Lego-like building blocks, the different optimal reaction conditions for individual enzyme, and the use of costly labile coenzymes. 目前无细胞生物转化合成途径的制约条件包括缺少像Lego的组成形式的稳定的酶，不同的酶具有不同的最佳反应条件，以及昂贵的不稳定辅酶的使用。It is expected that high-yield SyPaB will be an important platform for producing low-cost biofuels and biochemicals.不难想象，高收率无细胞生物转化合成途径（SyPaB）将是生产低成本的生物燃料和物品的重要途径。IntroductionSustainable development of humankind needs the production of renewable energy at affordable costs. 人类的可持续发展需要用可以承受的代价生产可再生能源。Current transportation accounts for approximately 20% of global energy consumption. 目前，全球约20的能源消耗在交通运输上。Owing to some special requirements for the transportation sector, such as high power density and high-energy storage capacity in a small volume, potential solutions for the transportation sector are limited. 由于对交通部门的一些特殊要求，如高功率密度和高能量小体积的存储容量，很少存在解决运输部门可能的方案。Biomass, the most abundant renewable bioresource, is the only low-cost renewable resource that can be used for the production of large amounts of transportation fuels and renewable materials (e.g., paper and polylactic acid). 生物是最丰富的可再生生物资源，也是唯一低成本的可用于运输燃料和大量生产再生材料（例如，纸和聚乳酸）的可再生资源， Natural terrestrial plants collect nonpoint intermittent low-energy-flux solar energy and store it in the form of low-cost chemical energy biomass that can be stored, distributed, and utilized easily and economically. 自然界中的陆地植物收集非点间歇性低能量通量的太阳能，并将简单的经济性的其以低成本的化学能生物量的形式储存储存、分布、利用。Starting from biomass sugars, numerous biocatalytic or catalytic approaches have been proposed and investigated for the production of various biofuels, such as ethanol 1, high-chain alcohols 2, alkanes 3, fatty acids or their esters 4,5, hydrogen 6_,7_, methane 8, and hydrocarbons 9. 人们已经研究了用生物质糖、生物催化或催化方法合成各种生物燃料，如乙醇，高链醇，烷烃，其脂肪酸或酯，氢，甲烷和碳氢化合物。Liquid biofuels work well with current infrastructure and internal combustion engines, representing a short-term or middle-term solution to the transportation sector. 液体生物燃料在现在的基础设施和内燃机中可以很好的应用，已经可以很好的解决中短距离的运输。In future, hydrogen fuel cell-motor system represents a long-term solution for the transportation sector because hydrogen fuel cells have higher energy efficiencies and produce less pollution than internal combustion engines. 在未来，氢燃料电池发动机系统是一个为运输部门的长期解决方案，因为氢燃料电池具有更高的能源效率和具有比内燃机有更少的污染。Hydrogen can also be produced from diverse primary energy sources . 氢还可以从不同的初级能源生产。Cell-free synthetic pathway biotransformation (SyPaB) is the in vitro assembly of a number of (purified) enzymes and coenzymes for mediating complicated biochemical reactions 11_,12_. 无细胞生物转化合成途径（SyPaB）在体外装配了复杂的生物化学反应中（纯化）的酶和辅酶。Cell-free SyPaB, as one direction of synthetic biology, has a number of potential advantages over fermentations mediated by intact microbes. 无细胞生物转化合成途径（SyPaB）作为合成生物学的一个分支，比以完整的微生物的发酵形式具有潜在的优势。These advantages include high product yield, fast reaction rate, high product titer, broad range of reaction conditions, and simplified process control.这些优势包括产品的高产量，反应速度快，产品的高精度，较宽的反应条件选择性，并可以使控制过程简化。The development cycle of SyPaB involves five parts: first, pathway reconstruction, second, enzyme selection, third, enzyme engineering, fourth, enzyme production, and fifth, process engineering. 对SyPaB开发周期包括五个部分：一，通路重建，第二，酶的选择，第三，酶工程，第四，酶的生产，以及第五，过程工程。Furthermore, it is vital to construct in vitro enzyme complexes for minimizing degradation of labile metabolites and facilitating metabolite channeling for some cascade enzymatic reactions 13. 此外，酶复合体在体外构建至关重要的是尽量减少不稳定的代谢产物和促进代谢降解某些级联酶促反应。Living entities, such as microbial cells, can be engineered to produce desired products by in vivo synthetic biology technology. 人们可以利用活的微生物，如微生物活细胞，在体内合成所需的产品。Achievement of high product yield through this means, however, requires labor-intensive and timeconsuming optimization of complex cellular networks. 但是，通过这种方法合成产品的产量高的代价是需要密集的劳力，耗时复杂的蜂窝状网络的优化。By contrast, high product yield can be achieved more efficiently with in vitro systems when system components are available 6_,7_. 相比之下，当反应系统可以获得时，在体外系统时中合成高产率产品更有效。Figure 1 compares biocatalysis complexity of in vitro and in vivo platforms. 图1比较了在体外和体内复杂的生物催化反应。Taking a relatively simple pathway involving six cascade biochemical reactions where each step has five choices (genes or enzymes), in vitro systems would have 30 combinations since each enzymatic step can be easily exchanged by another enzyme. 以一个相对简单的包括六个生化级联反应的途径为例，其中每个步骤都有五个选择（基因或酶），在体外系统将有30个组合方式，因为每个步骤中的酶都可以很容易被另一酶代替。In vivo systems may have 56 = 625 combinations because each enzymatic step is linked with others. 在体内系统可能有56 = 625的组合，因为每一步酶与其他的联系在一起。Furthermore, in vivo systems are complicated by the possibility that reaction rate at each step in the enzymatic pathway involves regulation at the level of gene transcription, mRNA stability, and translation. 此外，在体内系统的复杂反应的可能性，在每一个步骤的酶通路率涉及在基因转录，mRNA的稳定性，和翻译水平调节。They may also be regulated by processes involved in delivery of the gene product to the site of function in the cell as well as proteinprotein and proteincofactor interactions. 他们还可能受某些过程的调节。这些过程发生在细胞中把基因产品传递到特定的功能部位和蛋白质-蛋白质和蛋白质-辅因子的相互作用。In this article, we present the advances in SyPaB for biofuel production, discuss SyPaB advantages and limitations and offer suggestions for R&D priorities in the future.在这篇文章中，我们提出在无细胞生物转化合成途径（SyPaB）用于生产生物燃料的进展，讨论无细胞生物转化合成途径（SyPaB）优势和局限性，并为今后的研发重点提出建议。Production of biofuels生物燃料的生产Synthetic pathway assembly is usually designed based on natural metabolic pathways with necessary modifications with ATP and coenzyme balanced. 组合的合成途径通常基于自然代谢途径的必要的修改。这些修改是关于ATP和辅酶平衡的。Since biofuels are low-value products, it is economically prohibitive to produce them using expensive feedstock.由于生物燃料是低附加值产品，在经济上它是不允许使用昂贵的生产原料的。Alcohols乙醇A high octane liquid fuel blend of ethanol can be produced through microbial anaerobic fermentation. 乙醇的高辛烷值的液体混合燃料可通过微生物厌氧发酵生产。The Nobel Prize winner Eduard Buchner discovered ethanol fermentation by a cell-free yeast extract in 1897 17. 诺贝尔文学奖获得者Eduard Buchner在1897年发现一无细胞酵母发酵乙醇提取物。Net ATP accumulation (i.e., 2 ATP produced per glucose), however, prevents complete conversion of glucose to ethanol (Eqn (1):但是，ATP的净积累（即每葡萄糖生产2 ATP）阻止葡萄糖完全的转化为乙醇（式（1）： (1)Welch and Scopes 18 demonstrated the feasibility of high-yield ethanol production by a reconstituted yeast glycolytic enzyme system containing ATPase, which facilitates complete conversion of 180 g/L glucose to 90 g/L ethanol within eight hours with 99% of the theoretical yield. Welch and Scopes证明了一个含有ATP酶的重组酵母糖酵解酶系统生产高产量的乙醇产品的可行性。它可以在八小时之内把180g/ L葡萄糖转化成90 g / L乙醇，这种转换可以达到99的理论产率。Alternatively, substitution of arsenate for inorganic phosphate can constrain ATP synthesis and prevent ATP accumulation in this reaction 19. 另外，无机磷酸盐的替代物砷酸盐可以限制ATP的合成，并防止在这个反应中ATP的积累。The enzymes that require phosphate can use arsenate because arsenate is structurally and chemically similar to phosphate. 需要磷酸盐的酶可以用砷酸盐代替的原因是砷酸盐和磷酸盐的结构和化学性质类似。Since organic compounds of arsenate are less stable than the analogue phosphate compounds, they decompose rapidly by spontaneous hydrolysis, resulting in dissipation of high-energy phosphate bonds 19. 由于有机砷化合物的稳定性不如磷酸盐化合物的类似物，他们自发地迅速地发生水解，这些会导致高能磷酸键的消耗。Another fuel that can be produced biologically is butanol. Butanol has a higher energy density and more hydrophobicity than ethanol, but it cannot be produced at as high a yield and titer as ethanol 20. 另一个可以生产生物燃料的是丁醇。丁醇比乙醇具有更高的能量密度和更好的疏水性，但它不能和乙醇一样具有高产量和浓度。Cell-free SyPaB butanol production may have high product yield, produce high product titer, simplify process control, and have no risk of bacteriophage contamination 11_. 无细胞生物转化合成途径（SyPaB）的丁醇生产可能有较高的产品产量和高滴定度，并可以简化过程控制，且无噬菌体污染风险。Because two moles of ATP are produced and accumulate per mole of glucose in the natural butanol pathway (Eqn (2), substitution of arsenate for inorganic phosphate in the enzyme mixture or addition of ATPase to degrade the ATP can prevent ATP accumulation in this reaction 19:在自然丁醇途径（式（2）中，每摩尔葡萄糖有两摩尔的ATP生产和积累，在酶的混合物中用无机磷酸盐的替代物砷酸盐或者在ATP中加入ATP酶进行降解可以防止在这一反应中ATP积累： (2)Hydrogen氢气Carbon-neutral hydrogen gas is the preferred energy carrier for the future transportation sector. 碳中和氢气是未来运输部门首选的能源载体。Hydrogen can be produced from biomass sugars through numerous methods employing chemical catalysis, biocatalysis, or a combination of both. 氢气可以通过从生物量的糖用多种方法进行生产。这些方法包括化学催化方法，生物催化方法，或者是两者结合的方法。However, most of these technologies suffer from low hydrogen yield (far below the theoretical yield = 12 H2 per glucose), undesired products, and/or severe reaction conditions 10_. 然而，这些技术大多都有低氢产量（远低于理论收益率= 每摩尔葡萄糖生产12 摩尔的H2），副产品和/或苛刻的反应条件10_。These cell-free synthetic enzymatic pathways generate nearly 12 moles of hydrogen per mole of glucose unit of starch or cellulosic materials 6_,7_:在这些无细胞合成酶途径中，每单位淀粉或纤维素葡萄糖可以产生近12单位氢： (3)These synthetic pathways contain: first, a chain-shortening phosphorylation reaction for producing glucose-1- phosphate (G-1-P) catalyzed by glucan phosphorylase ;second, conversion of G-1-P to glucose-6-phosphate (G-6-P) catalyzed by phosphoglucomutase; third, a pentose phosphate pathway containing 10 enzymes for producing 12 NADPH per G-6-P, and fourth hydrogen generation from NADPH catalyzed by hydrogenase. 这些合成途径包括：第一，生产1 -磷酸-葡萄糖（G - 1 - P）。这属于链截短的磷酸化反应，其中的催化酶是葡聚糖磷酸化酶；第二，1 -磷酸-葡萄糖（G - 1 - P）转化为6-磷酸-葡萄糖（G-6-P）。其中的催化剂为磷酸葡萄糖变构酶；第三，戊糖-磷酸途径，每单位的G - 6 P可以生产12个单位的NADPH。这步需要十种酶进行催化。；第四，NADPH通过氢化酶产生氢。For the first time, these endothermic entropy-driven biochemical reactions achieve a chemical energy output (hydrogen)/input (carbohydrate) = 1.22 by absorbing ambient temperature waste heat 6_,7_. 这些吸热熵驱动的生物化学反应能够实现化学能输出（氢）/输入（碳水化合物）是第一次，。此反应输出和输入的能量比为1.22。这个反应的发生是通过吸收环境中的余热。Since glucan phosphorylases are responsible for generating G-1-P in these pathways, the yield of G-1-P is (n _ 1)/n, where n is the degree of polymerization of oligosaccharides or polysaccharides. 在这些途径中，葡聚糖磷酸化酶是用来产生的G - 1 - P的，对G - 1 - P的收益率是（n - 1）/ n，其中n是寡糖或多糖聚合度。Therefore, it is vital to identify the enzymes that can hydrolyze long chain insoluble cellulose for the generation of soluble G-6-P without use of costly ATP.因此，至关重要的是要确定没有酶能水解不可溶性的纤维素到可溶性可溶性的G - 6 P。而这种酶不需要消耗昂贵的ATP。Polyols alkane precursors多元醇-烷前体Long chain alkanes made from biomass sugars are a sulfur-free liquid fuel that can be used for jet planes and heavy-duty vehicles 21. 生物量糖类生产的长链烷烃是一种无硫液体燃料，这种无硫的液体燃料可用于喷气飞机和重型车辆使用。Recently, we have developed a new approach to produce alkane precursors polyols (e.g., xylitol and sorbitol) from biomass sugars by SyPaB (submitted). 最近，我们已经开发出一种通过SyPaB的新的方法来生产烷烃的前体-多元醇（如木糖醇，山梨醇）。For the production of alkanes, aqueous-phase reforming (APR), chemical reforming occurring in aqueous phase under modest temperature and high pressure conditions, can convert water-soluble polyols to water-immiscible alkanes 3,22,23. 对于烃类的生产，我们使用水相重整（APR）技术。即在温和的温度，高压条件下发生的水相中的发生化学重整反应，可以将水溶性多元醇转变为水不溶烷烃。This combination of biocatalysis and chemical catalysis has several advantages: a very high-energy retraining efficiency from sugars to alkanes (95%, overall; 99.6%, SyPaB; 95%, APR), high yield and low cost for xylitol and sorbitol generation, tolerance to the dilute acid-pretreated biomass hydrolysate that inhibits microorganism growth and metabolisms, modest reaction conditions, and low product separation costs. 这种生物催化和化学催化组合有几个优势：从糖到烃类的非常高的能量效率（整体，95; SyPaB，99.6，APR，95），高产量和木糖醇和山梨醇的低的成本，抑制微生物的生长和新陈代谢的耐稀酸水解生物预处理，温和的反应条件，低的产品分离成本。This new pathway contains 12 enzymes similar to the cellobiose (C12H22O11)-to-hydrogen enzyme cocktail7_ but without hydrogenase. 这个新的途径包含12个类似的纤维二糖-氢酶但没有氢。The latter is replaced by an aldose reductase (EC 1.1.1.21) 24, whereby 1 mole of cellobiose can produce 12 NADPH and one glucose (Eqn (4):后者可以用一种醛糖还原酶（EC1.1.1.21）代替，借助于此，1摩尔的纤维二糖可以生产12摩尔 NADPH和1摩尔葡萄糖（式（4）： (4)The overall reactions for xylitol (xylitol) and sorbitol (sorbitol) synthesis from xylose and glucose plus a small amount of cellobiose are从木糖和葡萄糖到木糖醇（木糖）和山梨醇（山梨醇）的综合反应体系反应如果加上少量的纤维二糖，则为 (5) (6)The conversion of xylitol and sorbitol to alkanes by APR is a weakly exothermic process 21,22,25:木糖醇和山梨醇的通过APR方法转化为烷烃是一种弱放热过程： (7)The products of alkanes have only 30% of the mass of polyols while retaining approximately 95% of the combustion energy in the reactants. 链烷烃产品只有30的多元醇，同时在反应物中保留的燃烧能量大约占95。In addition, separation of water-insoluble alkanes with water is far less costly than that of alcohols and water. Longer chain alkanes from C7 to C15 can be produced by combining the aqueous-phase dehydration/hydrogenation process with a CC bond-forming aqueous-phase aldol condensation step 21. 此外，不溶于水的正构烷烃与水的分离比起酒精和水的分离来说，会有很低的价格。C7到C15的长链烷烃可以与水相脱水/加氢相结合，与C - C键形成水相一步缩合加氢过程。Weight-based total turn-over number 以质量为基础的Kcat值A major difference between microbial fermentation and SyPaB is that microbes can duplicate themselves whereas enzymes cannot. 微生物发酵和SyPaB的主要区别是微生物可以复制，繁殖，而酶却不能。Because SyPaB requires enzyme production by microbial fermentation, purification, and stabilization of enzymes as well as the addition of costly coenzymes, it is plausible that SyPaB is more costly than microbial fermentation. 由于SyPaB需要由微生物发酵，纯化和固定酶，以及需要添加昂贵的辅酶，所以认为SyPaB比微生物发酵昂贵貌似是非常合理的。Therefore, we suggest that comparison of microbial fermentation and SyPaB must be based on weight-based total turn-over number (TTNW) in terms of kg of product per kg of biocatalyst: TTNW product biocatalyst (8) 因此，我们认为，微生物发酵和SyPaB比较应当是根据每千克的生物催化剂可以生产多少千克的产品的TTNW来判定。TTNW=（8）As shown in Figure 2, self-duplicating living entity-based biotransformations have very low values of TTNW (kg product per kg biocatalyst). 如图2所示，可以自我复制的活的生物体的生物转化的TTNW值非常低。A typical ethanol batch fermentation has TTNW values of 36. 一个典型的乙醇发酵的TTNW值为3-6。However, when yeast cells are recycled (i.e., less cell mass is generated and more ethanol is produced), this operation leads to higher TTNW values (e.g., 1020) 27. 然而，当酵母细胞回收再利用（即很小的细胞总量产生更多的乙醇），此操作可以有更高的TTNW值（如10-20）。Typical TTNW values for intracellular recombinant protein production by microbial fermentations reactions range from 0.005 to 0.25 (i.e., _150% of cellular protein is target recombinant protein and total cellular proteins account for _50% of cellular weight). 由微生物发酵的细胞内重组蛋白生产值的TTNW范围从0.005到0.25（即目标重组蛋白占了细胞蛋白的大约1-50，细胞总蛋白约占细胞总重量的50）。For example, a recombinant protein Thermotoga maritime 6-phosphogluconate dehydrogenase (Tm 6PGDH) accounts for approximately 30% of the Escherichia coli cellular protein 28, resulting in a TTNW value of _0.15 (Figure 2). 例如，基因重组蛋白Tm 6-磷酸葡萄糖酸盐脱氢酶（Tm 6PGDH）约占大肠杆菌细胞蛋白30左右， TTNW值在0.15左右。Production of secretory proteins such as cellulase or protease has TTNW 1, that is, more cost-effective protein production. 分泌蛋白质如纤维素酶或蛋白酶的TTNW能够达到1以上，即，分泌蛋白质是更具成本效益的蛋白质。Biotransformations mediated by enzymes usually have far higher TTNW values than by living entities (Figure 2). 酶介导的生物转化的通常比微生物活体生产有更高的TTNW值。Three thermoenzymes from our laboratory exhibit very high TTNW values: 415,000, Clostridiumthermocellum phosphoglucomutase29; 420,000, Tm 6PGDH 28; and _200,000, T. maritime fructose-1,6-bisphosphatase 13. 从我们的实验室获得的三种热酶表现出非常高的TTNW值：415000，梭菌的葡萄糖磷酸变构酶是415000，Tm 6-磷酸葡萄糖酸盐脱氢酶为420000，和Tm1,6-二磷酸果糖酶约为200000。Much higher TTNWvalues of these thermostable enzymes are expected after immobilization, like those of immobilized thermophilic glucose isomerase 30. Recently, we have obtained TTNw values of more than 5,000,000 for immobilized C. thermocellum phosphoglucose isomerase (unpublished). 像固定化后的嗜热的葡萄糖异构酶一样，这些耐高温酶固定化后TTNW会高得多。最近，我们获得了TTNw值超过500万的固定化了的C.thermocellum磷酸葡糖异构酶，但是还没有进行报道。Carbohydrate allocation to biofuels and biocatalysts 作为生物燃料和生物催化剂的碳水化合物Carbohydrate is used as both a carbon source to support microorganism growth for producing biofuel or producing enzymes and an energy source for biofuel production at the same time. 糖类是碳源，提供了微生物生存和产生生物燃料和酶所需的碳源。同时，它也可以作为生产生物燃料的能源物质We refer to the allocation of carbohydrate between biocatalysts (enzymes or microorganisms) and biofuels. 我们指的是生物催化剂（酶或微生物）和生物燃料所需的糖类。When a significant amount of carbohydrate feedstock is consumed for the synthesis of cell mass, it lowers biofuel practical yields. 当大量的碳水化合物做为微生物生长的原料时，生物燃料的实际产量就降低了。Since the carbohydrate costs account for more than 50% of the final prices of biofuels 11_,31, it is vital to estimate carbohydrate allocation (percentage) between the desired biofuel and biocatalysts (enzymes or microorganisms). 由于生物燃料成本中，糖的成本超过了50%，所以生物燃料和生物催化剂（酶或微生物）中糖类分配（百分比）是至关重要的。Figure 3 shows typical values of carbohydrate allocation to biocatalysts (AX/S): _50%, aerobic fermentations 32; _20%, microaerobic fermentations 33; _10%, anaerobic fermentations 34,35; and _25%, resting-cell biotransformation32. 图3显示了分配给生物催化剂（AX/S）典型值：好氧发酵为大约50， 微好氧发酵大约为20%厌氧发酵为大约10，静息细胞的生物转化大约为2% 到 5。These data suggest that it is not economically feasible to produce biofuels through aerobic