1、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
2、）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 biofue
3、ls 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
4、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。Intr
5、oductionSustainable 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 sect
6、or, 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 us
7、ed 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-cos
8、t 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
9、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, representi
10、ng 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 poll
11、ution 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
12、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
13、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, enzym
14、e 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 c
15、hanneling 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 t
16、his 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
17、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
18、 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 s
19、ystems 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 deliv
20、ery 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
21、 offer suggestions for Rsecond, 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（
22、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
23、 (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 pol
24、ymerization 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
25、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 approa
26、ch 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 hi
27、gh 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 t
28、o 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
29、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 ald
30、ose 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)+ +12 2612 2CHO NADP7HOC+NADPH 6COThe overall reactions for xylitol (xylitol) and sorbitol (sorbitol) synthesis f
31、rom 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)510610551222CHO + 7H2O !C +6O(6)6 64The 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)61461429CHO 3 +CO HThe
32、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 alk
33、anes 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 Kc
34、at 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
35、 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
36、 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）产 物 的 质 量生 物 催 化 剂 质 量As shown in Figure 2, self-duplicating living entity-based biotransformations have very low values of TTNW (kg product per kg biocatalyst).
37、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
38、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 weigh
39、t). 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 (Figu
40、re 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 hav
41、e 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
42、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 th
43、an 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 sourc
44、e 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
45、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 e
46、stimate 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%, micr
47、oaerobic 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 fermentations becaus
48、e of the high carbohydrate allocation to microorganism. 这些数据表明，利用好氧微生物发酵生产生物燃料在经济上是不可行的，因为微生物生长消耗了过多的糖类物质。Because carbohydrate has nearly constant costs ($0.18/kg carbohydrate = $11.1/GJ) 10_,36, it is important to evaluate any biofuel ($2040/GJ) produced from carbohydrate based on its mass yield an
49、d energy-retaining efficiency 37. 因为碳水化合物成本几乎不变（$ 0.18/kg 碳水化合物 = $ 11.1/GJ） ，评估生物燃料（$ 20-40/GJ）的可行性就由碳水化合物的产量和能源的最终剩余效率所决定。In SyPaB, carbohydrate allocation to the enzymes depends on TTNW and recombinant protein yield based on carbohydrate (YP/S, gram of protein per gram of carbohydrate consumed) (Figure 3b). 在 SyPaB 系统中，酶制剂所需的碳水化合物取决于 TTNW 和重组蛋白产量取决于碳水化合物（YP/ S，每克蛋白质消耗碳水化合物的重量） 。Higher YP/S results in lower carbohydrate allocation to enzyme synthesis, that is, more carbohydrate is allocat
