Future directions in cofactor research: Discovery and development of coenzyme F420-dependent bioprocesses

辅因子(辅酶)是辅助分子，对许多酶的催化活性至关重要。虽然一些辅因子在自然界中无处不在，但另一些辅因子存在于代谢高度专门化的微生物中。例如，辅酶F420在产甲烷微生物、抗生素产生菌以及结核病病原体中发挥着生理作用。依赖F420的酶对生物催化非常感兴趣，因为它们能够促进具有挑战性的化学反应。尽管有这些非凡的功能，F420仍然没有得到充分的探索，几乎没有在生物技术中使用。其中一个原因是辅因子的利用率很低，因为化学合成很麻烦，目前辅因子的微生物来源也不令人满意。因此，我们的目标是找出F420生物合成中缺失的一步，以便在模式菌大肠杆菌中生产它。然后，通过合理的基因工程和定向进化，生产效价将得到提高。为了有效地利用辅因子，需要一个再生系统，在反应后将辅因子重置到其原始状态。F420确实存在一些，然而，所有这些都受到了严重的限制。因此，我们将建立一个基于廉价基质的高效再生系统。这些努力将使F420对生物技术应用更具吸引力，并将促进进一步的工作包。特别是，我们的目标是确定具有潜在生物技术用途的依赖F420的新生物过程。我们的初步结果证实了最近的生物信息学研究，这些研究表明F420依赖的酶比预期的更广泛。利用它们与F420结合的能力，这些酶将从各种细菌中得到浓缩，并进行学术上耐人寻味的测试和生物技术上的有用活动。此外，我们还将研究异常生态系统中的F420。为此，我们将解决细菌和霉菌之间的共生问题，霉菌是水稻苗枯病的病原体。我们的初步结果表明，F420是在共生体与其真菌宿主生活在一起时产生的。在合作中，我们想要阐明F420在这个模式细菌-真菌联盟中的功能，这对微生物生态学和植物病理学具有非常重要的意义。

Cofactors (coenzymes) are helper molecules that are crucial for the catalytic activity of many enzymes. While some cofactors are ubiquitous in nature, others are found in microbes with a highly-specialized metabolism. Coenzyme F420, for instance, plays a physiological role in methanogenic microbes, antibiotic-producing bacteria, as well as in the tuberculosis pathogen. F420-dependent enzymes are highly interesting for biocatalysis, since they are able to promote challenging chemical reactions. Despite these extraordinary features, F420 remains underexplored and barely used in biotechnology. One reason for this is the low availability of the cofactor as chemical synthesis is cumbersome and microbial sources of the cofactor are non-satisfactory at the moment. Therefore, our goal is to identify a missing step in the biosynthesis of F420 to produce it in the model bacterium Escherichia coli. Production titers will then be increased by rational genetic engineering as well as directed evolution. To effectively use cofactors, a regeneration system is required that resets the cofactor to its original state after reaction. Some do exist for F420, however, all of them suffer from severe limitations. Thus, we will establish an efficient regeneration system based on inexpensive substrates. These efforts will render F420 more attractive for biotechnological applications and will facilitate further work packages.In particular, we aim to identify novel F420-dependent bioprocesses with potential use for biotechnology. Our preliminary results corroborate recent bioinformatics studies that suggest that F420-dependent enzymes are more widespread than anticipated. Exploiting their ability to bind to F420, these enzymes will be enriched from various bacteria and tested for academically intriguing and biotechnologically useful activities. Additionally, we will investigate F420 in unusual ecological systems. To this end, we will address the symbiosis between bacteria and a mold fungus, the causative agent of rice seedling blight. Our preliminary results demonstrate that F420 is produced as soon as symbionts live in association with their fungal host. In collaboration, we want to elucidate the function of F420 in this model bacterial-fungal alliance that is of high importance for microbial ecology and plant pathology.