Growth-associated gene essentiality in Streptomyces coelicolor.

链霉菌具有大量的特征，这些特征在深层培养的快速生长过程中可能是多余的。这包括形态分化和次生代谢(例如抗生素生产)。尽管如此，链霉菌发酵罐培养物被用于生产许多生物制品，在这些过程中，快速、高密度的生长至关重要。许多实验室正试图确定能够提高链霉菌在发酵罐中的性能的特征，从而定义能够高效合成新产品的超级宿主菌株的特征。天蓝色链霉菌是该属中第一个被测序的物种，对其分子生物学的了解比其他任何物种都多，因此它有可能成为超级宿主。然而，许多人发现在发酵培养中很难快速生长。这些想法形成了我们对了解不同基因的需求如何随着生长速度的变化而变化的兴趣的背景。我们对编码酶的基因特别感兴趣，这些基因是最近发表的S天蓝色基因组规模代谢网络的一部分，因为我们可以进行计算机建模，预测网络中代表的每个基因的缺失对生长速度的影响。因此，基因本质上是模型研究中的一个相对术语，根据基因缺失对生长速度的影响，某些基因比其他基因更重要。在这项研究中，我们计划将这一定量重要性概念扩展到活体实验中，将两种基因重要性测量方案中的一种应用于以不同速度生长的细胞。因此，我们将确定哪些基因在每种生长速度下是必不可少的，并将这些发现与计算机预测进行比较。我们也有改进现有预测技术的想法，这个项目的成功结果将使我们能够第一次用实验室实验验证预测。我们将把那些看起来与生长速度相关的必要基因与那些在快速生长的菌株中表达的基因进行比较，这些菌株是在恒化器中选择的，恒化器是一种迫使细胞以我们指定的速度生长的设备。我们还将迫使亲本菌株以不同的速度生长，这样我们就可以确定表达对生长速度反应的基因是否相同，其重要性随着生长速度的不同而变化。我们的体内基因重要性实验，使用现有的技术，涉及在琼脂上电镀一组突变体，其中每一种可能的基因敲除都代表着，然后我们将它们放入恒化器中，以确定哪些突变体可以在恒化器中以各种生长速度存活下来。可以说，最初的培养步骤本身是有选择性的，因为只有那些能够在平板上形成集落的细胞才能进入恒化器。这可能是不可避免的，不应不适当地削弱我们研究结果的有效性。然而，我们也计划了一种以前从未在链霉菌中尝试过的方法，这种方法不会允许基因沉默突变本身出现，直到突变体在迫使它们以每个指定的速度生长的条件下处于恒化器中。如果我们能让这种新方法奏效(我们有一个团队，其广博的经验让我们有信心我们会做到)，那么它就会被使用。如果不是，我们将求助于更传统的方法。

Streptomyces species have a large number of characteristics that are, presumably, redundant during rapid growth in submerged culture. These include morphological differentiation and secondary metabolism (e.g. antibiotic production). Despite this, Streptomyces fermenter cultures are used for the production of many bioproducts, in processes in which rapid, high density growth is of paramount importance. Many laboratories are trying to identify characteristics that would enhance the performance of Streptomyces species in fermenters, thereby defining the characteristics of a 'superhost' strain, capable of synthesising new products, with high efficiency. Streptomyces coelicolor was the first species in the genus to be sequenced and more is known about its molecular biology than that of any other species, so it is potentially a good candidate for development into a superhost. However, many find it very difficult to grow at rapid rates in fermenter culture. These ideas form the background to our interest in knowing how the requirements for different genes change as a function of growth rate. We are particularly interested in genes coding for enzymes that are part of the recently-published S coelicolor genome scale metabolic network, as we can carry out computer modelling that predicts the effect of deletion of each gene, represented in the network, on growth rate. Gene essentially is therefore, a relative term in modelling studies, some genes being 'more essential than others' depending on the effect that their deletion has on the growth rate. In this study, we plan to extend this quantitative essentiality concept to in vivo experimentation, with the application of one of two gene essentiality measurement protocols to cells growing at different rates. Thus we will determine which genes are essential at each growth rate and compare these findings to the computer predictions. We also have ideas for improving on existing prediction techniques and, a successful outcome to this project will enable us to validate the predictions with laboratory experiments, for the first time. We will compare those genes that seem to have growth rate related essentiality to those that are expressed in a rapidly growing strain, selected in a chemostat, a device that forces cells to grow at the rate that we specify. We will also force the parental strain to grow at different rates, using this apparatus, so that we can determine whether the genes whose expression responds to growth rate, are the same genes whose essentiality varies with the growth rate. Our in vivo gene essentiality experiment, using existing techniques, involves plating a population of mutants, in which every possible gene knock-out is represented, onto agar before we put them in the chemostat to determine which mutants can survive in the chemostat at each of a number of growth rates. It could be argued that this initial plating step is, itself, selective as only those capable of forming colonies on plates will make it into the chemostat. It may be that this will be inevitable, and should not unduly detract from the validity of our findings. However, we also plan an approach, never previously attempted in Streptomyces, that will not allow gene silencing mutations to manifest themselves, until the mutants are in the chemostat under the conditions that force them to grow at each specified rate. If we can make this new approach work (we have a team whose breadth of experience give us confidence that we will) then it will be used. If not, we will fall back on the more conventional approach.