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越来越多的液体燃料消费。尽管考虑到目前的石油价格，这很有吸引力，但主要的驱动力是根据《京都议定书》做出的减少温室气体排放的承诺。与化石燃料不同，从绿色植物中提取的燃料实际上是二氧化碳中性的。由糖发酵生产的乙醇是一种成熟的生物燃料，在巴西和美国已经得到广泛使用，而且已经有了使用纯乙醇或乙醇-石油混合物来驱动汽车的技术。生产乙醇的经典方法是使用酵母发酵蔗糖(从甘蔗或甜菜中提取)或葡萄糖(从淀粉中提取)。酵母是少数几种能够将糖专门发酵为乙醇和二氧化碳的生物之一，它通过使用丙酮酸脱羧酶(PDC)和酒精脱氢酶(ADH)来做到这一点。细菌中很少发现PDC，这是酿造行业以及最近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乎其微的，显然，如果有可能利用生物质中存在的所有糖，特别是半纤维素和纤维素中的那些糖，这一点和整体经济将大大改善，这两种糖共同构成了全球最丰富的碳水化合物来源。不幸的是，面包师/啤酒酿造商的酵母不会自然发酵半纤维素中的戊糖(C5)，而那些能够发酵的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的典型顺序批处理过程更有效率，后者在运行之间会产生显著的“死时间”。连续过程意味着连续去除乙醇，这是在高温(乙醇的沸点为78摄氏度)下操作最有效的方法。因此，生产乙醇的理想生物应该在70oC左右的温度下快速发酵各种糖，包括戊糖，可能还有更复杂的底物，如纤维素(在这种温度下，可以使用气体汽提去除乙醇)。然而，这样的微生物还没有被分离出来。酵母在50摄氏度以上的温度下不能生长，而经常能代谢一系列糖(包括复杂的聚合物)的嗜热菌往往会产生多种发酵产物，其组成可能取决于生长条件。这项建议提出了两种构建嗜热菌的策略，这些细菌专门从一系列生物质衍生的糖中生产乙醇。它的前提是，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转化为乙醇生产将比寻找和设计一个良好的嗜热乙醇生产商来使用广泛的底物更可行。第一种策略是使用“定向进化”的方法来生产一种改良的PDC，它可以在65-70摄氏度的嗜热菌中工作。这主要包括高频突变相关基因，和/或重组存在于嗜热菌中的已知相似基因的元素，并结合一种强大的改良变种选择方法。第二种策略涉及基于已知在嗜热菌中表达的酶的组合创建一种新的发酵途径，但这些酶通常不在一起表达。特别是，它涉及到在厌氧条件下表达丙酮酸脱氢酶，这是一种通常与有氧生长有关的酶，以及两种通常的厌氧酶。总而言之，这些途径将产生与PDC途径相同的结果。我们有过这样的先例，即这个途径已经在嗜热葡萄糖苷酸菌的突变体中发挥作用。

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.