Microorganisms used as “production plants” for active ingredients are an important asset in the sustainable bioeconomy toolkit. But how can production strains be manipulated most efficiently for optimal performance in the process? A new approach is based on miniaturization and automation.
In the transformation from conventional, oil-based industry to a sustainable bioeconomy, greater reliance will be placed on microorganisms for the production of active ingredients made from sustainable resources. More efficient approaches to strain construction will be needed to meet future challenges in the development of platform organisms. To meet this need, one of the technologies under development at the Microbial Bioprocess Lab, a Helmholtz Innovation Lab, is a miniaturized, automated process to support adaptive lab evolution.
The majority of microbial production processes which are relevant for industrial use are currently developed in three inter-dependent stages. The first step is to select a suitable production organism from a list of established microbial “workhorses”. The genetic basis for efficient, directed strain construction of these so-called platform organisms already exits. The list includes Escherichia coli, Saccharomyces cerevisiae, Bacillus subtilis and Corynebacterium glutamicum [1-4]. After that, the desired metabolic properties for production of the target components are systematically inserted into the selected organism, and the resulting strain variants are pre-sorted using fast screening techniques . Potential candidates are then characterized in detail. This involves the use of new techniques for fast strain phenotyping  combined in the most efficient way with multiomics analysis  and integrative modeling .
The last step is development of a production process. It is highly beneficial to specify clearly defined cultivation conditions, because that makes process optimization much easier later on to maintain long-term production stability. However, this three-stage approach generally results in long development cycles, particularly for the discovery of suitable “adjustment levers” in the target organism's genome. During metabolic debottlenecking, the enormous complexity of metabolism in the microorganism, which is the result of evolutionary adaptation to changing environmental conditions, creates a major hurdle. Not only that, genetic modifications derived from an independent, empirical or theoretical foundation often produce a large unintended reduction in the cellular fitness of the target organism. This can have a significant adverse effect on production output.
Adaptive lab evolution
Fast adaptation of microorganisms to different environmental conditions forms the basis for the adaptive lab evolution approach (ALE). Microorganisms are cultivated under non-natural environmental conditions which are process-relevant in order to improve growth behavior as a result of evolutionary adaptation. This includes in particular adaptation to specific operating conditions (temperature, pH, exposure to gas, etc.) and individual (toxic) media components as well as to intra- and extra-cellular (by) products produced by the cells.
Particularly with production processes which are coupled to microbial growth and involve alternative carbon sources that are often produced when green biomass is used (e.g. C5 sugars such as D-xylose) or when inhibiting target products (e.g. alcohols or organic acids) are formed, an expansion of the substrate spectrum or the product tolerance is a desirable end condition in ALE-based strain development .
During adaptation to the selected stress conditions, mutations in specific areas of the target organism’s genome occur more frequently. In contrast to directed strain construction, with the ALE technique there is no risk that cellular fitness will be reduced. Ideally, mutations which increase fitness will be assertive. Looking at the regulatory aspects, it should be noted that strain variants produced exclusively with ALE are no genetically modified organisms (non-GMO). Besides the direct use of an evolved strain for production purposes, gene sequencing can be used in parallel to identify the resulting mutations to form the basis for directed strain development .
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