Developing strategies and a toolbox for metabolic engineering of thermophiles for ethanol production

英国致力于用生物衍生燃料取代目前液体燃料消费的越来越多的部分。虽然考虑到目前的石油价格，这是有吸引力的，但其主要驱动力是在京都议定书下做出的减少温室气体排放的承诺。与化石燃料不同，那些来自绿色植物的燃料实际上是二氧化碳中性的。由糖发酵产生的乙醇是一种成熟的生物燃料，已经在巴西和美国广泛使用，并且存在纯乙醇或乙醇-石油混合物驱动汽车的技术。乙醇生产的经典方法使用酵母发酵蔗糖（来自甘蔗或甜菜）或葡萄糖（来自淀粉）。酵母是少数几种可以将糖专门发酵成乙醇和二氧化碳的生物之一，它通过使用丙酮酸脱羧酶（PDC）和乙醇脱氢酶（ADH）来实现。PDC很少在细菌中发现，这是酿酒业和最近的燃料乙醇生产使用酵母的主要原因。然而，从蔗糖和淀粉生产乙醇的能量平衡是微不足道的，并且很明显，如果可以使用生物质中存在的所有糖，特别是半纤维素和纤维素中可获得的那些糖，这一点以及整体经济性将得到很大改善，半纤维素和纤维素一起构成全球最丰富的碳水化合物来源。不幸的是，面包/啤酒酵母不会自然发酵半纤维素中发现的戊糖（C5），而那些酵母菌株生长非常缓慢。此外，运行连续发酵过程将比工业酵母发酵典型的连续分批过程更有效，连续分批过程在运行之间引起显著的“停滞时间”。连续工艺意味着连续去除乙醇，在高温下操作（乙醇的沸点为78 ℃）最有效。因此，一个理想的乙醇生产微生物将在70 ℃左右的温度下快速发酵各种糖，包括戊糖，以及可能更复杂的底物，如纤维素（乙醇可以在这个温度下使用气提去除）。然而，这样的生物体还没有被分离出来。酵母菌在50 ℃以上的温度下不能生长，而嗜热菌通常可以代谢一系列糖，包括复杂的聚合物，往往会产生多种发酵产物，其组成可能取决于生长条件。该建议提出了两种策略，用于构建专门从一系列生物质衍生的糖生产乙醇的嗜热细菌。它从这样的前提开始，即考虑到可能使用的不同生物质底物和预处理的范围，分离能够在各种底物上生长的细菌并将其下游代谢工程化到乙醇生产，比找到并工程化良好的嗜热乙醇生产者以使用广泛的底物更可行。第一种策略是使用“定向进化”方法来产生在65- 70 ℃的嗜热菌中工作的改性PDC。这基本上涉及以高频率突变相关基因，和/或重组来自嗜热菌中存在的已知相似基因的元件，结合用于改进变体的强大选择方法。第二种策略涉及基于已知在嗜热菌中表达但通常不一起表达的酶的组合来创建新的发酵途径。特别地，它涉及在厌氧条件下表达丙酮酸脱氢酶，一种通常与需氧生长相关的酶，以及两种通常厌氧的酶。总之，这些将具有与PDC途径相同的结果。我们有一个先例，这个途径已经在嗜热土芽孢杆菌的突变体中运作。

The UK is committed to replacing an increasing fraction of current liquid fuel consumption with biologically derived fuels. While this is attractive given the current price of oil, the primary driver for this was a commitment made under the Kyoto protocol, to reduce greenhouse gas emissions. Unlike fossil fuels, those derived from green plants are virtually carbon dioxide neutral. Ethanol, produced by the fermentation of sugars, is an established biofuel which is already extensively used in Brazil and in the USA, and the technology to run cars on either pure ethanol or ethanol-petroleum mixtures exists. The classic method for ethanol production uses yeast to ferment either sucrose (from sugar cane or beet) or glucose (from starch). Yeast is one of the few organisms that can ferment sugars exclusively to ethanol and carbon dioxide, which it does by employing the enzymes pyruvate decarboxylase (PDC) and alcohol dehydrogenase (ADH). PDC is rarely found in bacteria, which is the main reason why the brewing industry, and more recently, fuel ethanol production have used yeast. However, the energy balance of ethanol production from sucrose and starch is marginal and it is clear that this, and the overall economics would be much improved if it was possible to use all of the sugars present in biomass, particularly those available in hemicellulose and cellulose, which together comprise the most abundant global sources of carbohydrates. Unfortunately, bakers/brewers yeast does not naturally ferment the pentose (C5) sugars found in hemicellulose, and those yeast strains that do, grow very slowly. Furthermore, it would be more efficient to run continuous fermentation processes than the sequential batch processes typical of industrial yeast fermentations, which incur significant 'dead time' between runs. A continuous process implies continuous removal of ethanol, which is most efficiently achieved by operating at elevated temperature (the boiling point of ethanol is 78oC). Thus, an ideal organism for ethanol production would rapidly ferment a wide range of sugars, including pentoses, and possibly more complex substrates such as cellulose, at temperatures around 70oC (ethanol can be removed at this temperature using gas stripping). However, such an organism has not been isolated yet. Yeasts do not grow at temperatures above 50oC, while thermophilic bacteria that can often metabolise a range of sugars, including complex polymers, tend to produce multiple fermentation products, the composition of which may depend on growth conditions. This proposal presents two strategies for constructing thermophilic bacteria which produce ethanol exclusively from a range of biomass-derived sugars. It starts from the premise that, given the range of different biomass substrates and pretreatments that are likely to be used, it would be more feasible to isolate bacteria able to grow on the various substrates and engineer their downstream metabolism to ethanol production, than to find and engineer a good thermophilic ethanol producer to use a wide range of substrates. The first strategy is to use a 'directed evolution' approach to produce a modified PDC which works in a thermophile at 65-70oC. This essentially involves mutating the relevant gene at high frequency, and/or recombining elements from known similar genes present in thermophiles, combined with a powerful selection method for improved variants. The second strategy involves creating a novel fermentation pathway based on combinations of enzymes known to be expressed in thermophiles, but which are not normally expressed together. In particular it involves expressing pyruvate dehydrogenase, an enzyme usually associated with aerobic growth, under anaerobic conditions, together with two normally anaerobic enzymes. Together, these would have the same outcome as the PDC pathway. We have a precedent that this pathway already operates in mutants of the thermophile Geobacillus thermoglucosidasius.