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

开发用于乙醇生产的嗜热菌代谢工程的策略和工具箱

基本信息

批准号：
BB/E002994/1
负责人：
David Jonathan Leak
金额：
$ 43.08万
依托单位：
Imperial College London
依托单位国家：
英国
项目类别：
Research Grant
财政年份：
2007
资助国家：
英国
起止时间：
2007 至无数据
项目状态：
已结题

来源：
https://gtr.ukri.org/projects?ref=BB%2FE002994%2F1
关键词：
Developing strategies toolbox metabolic engineering

项目摘要

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.

英国致力于用生物学衍生的燃料代替当前液体燃料消耗的一部分。鉴于目前的石油价格，这很有吸引力，但这是根据京都协议做出的承诺，是减少温室气体排放的承诺。与化石燃料不同，从绿色植物中衍生的燃料实际上是二氧化碳中性的。乙醇是由糖的发酵生产的，是一种已建立的生物燃料，已经在巴西和美国广泛使用，并且在纯乙醇或乙醇 - 甲醇混合物上运行汽车的技术存在。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）。酵母是可以将糖专为乙醇和二氧化碳发酵的少数生物之一，它通过使用丙酮酸酶脱羧酶（PDC）和酒精脱氢酶（ADH）来做到这一点。 PDC很少在细菌中发现，这是为什么酿造行业以及最近的燃料乙醇生产使用酵母的主要原因。但是，蔗糖和淀粉产生乙醇的能量平衡是微不足道的，很明显，如果可以使用生物量中存在的所有糖，尤其是半纤维素和纤维素中的所有糖，这将大大改善，这将兼容最丰富的全球全球碳水化合物来源。不幸的是，面包师/酿酒师酵母并不自然发酵半纤维素中的五糖（C5）糖，而那些做得非常缓慢的酵母菌菌株。此外，运行连续发酵过程比工业酵母发酵的典型批量批量过程更有效，这在运行之间引起了显着的“死时间”。一个连续的过程意味着连续去除乙醇，这是通过在升高温度下运行（乙醇的沸点为78oC）最有效地实现的。因此，一种用于乙醇生产的理想生物会在70oC左右的温度下迅速发酵各种糖，包括五齿，可能是更复杂的底物（例如纤维素）（可以在此温度下使用气体剥离在此温度下除去乙醇）。但是，这种生物尚未孤立。酵母在50oC以上的温度下不会生长，而嗜热细菌通常可以代谢一系列糖，包括复杂聚合物，倾向于产生多种发酵产物，其组成可能取决于生长条件。该提案提出了两种构建嗜热细菌的策略，这些策略仅从一系列生物质衍生的糖中产生乙醇。从以下前提下，鉴于可能使用的不同生物量底物和预处理的范围，要分离能够在各种底物上生长的细菌并将其下游代谢的细菌隔离为乙醇的生产更为可行，而不是找到并设计出良好的热乙醇生产商来使用广泛的替代品范围。第一种策略是使用“定向进化”方法生产改良的PDC，该PDC在65-70oc的嗜热中起作用。这本质上涉及在高频中突变相关基因，和/或重组来自嗜热中的已知类似基因的元素，并结合了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剂中表达的酶的组合创建一种新颖的发酵途径，但通常不会一起表达。特别是它涉及表达丙酮酸脱氢酶，这是一种通常与有氧生长有关的酶，在厌氧条件下以及两种通常是厌氧酶。在一起，这些结果与PDC途径具有相同的结果。我们有一个先例，即该途径已经在热热葡萄糖酸的突变体中运行。