From frozen cell bank to product assay: high-throughput strain characterisation for autonomous Design-Build-Test-Learn cycles

Abstract

Background Modern genome editing enables rapid construction of genetic variants, which are further developed in Design-Build-Test-Learn cycles. To operate such cycles in high throughput, fully automated screening, including cultivation and analytics, is crucial in the Test phase. Here, we present the required steps to meet these demands, resulting in an automated microbioreactor platform that facilitates autonomous phenotyping from cryo culture to product assay. Results First, an automated deep freezer was integrated into the robotic platform to provide working cell banks at all times. A mobile cart allows flexible docking of the freezer to multiple platforms. Next, precultures were integrated within the microtiter plate for cultivation, resulting in highly reproducible main cultures as demonstrated for Corynebacterium glutamicum. To avoid manual exchange of microtiter plates after cultivation, two clean-in-place strategies were established and validated, resulting in restored sterile conditions within two hours. Combined with the previous steps, these changes enable a flexible start of experiments and greatly increase the walk-away time. Conclusions Overall, this work demonstrates the capability of our microbioreactor platform to perform autonomous, consecutive cultivation and phenotyping experiments. As highlighted in a case study of cutinase-secreting strains of C. glutamicum, the new procedure allows for flexible experimentation without human interaction while maintaining high reproducibility in early-stage screening processes.

Indoles and the advances in their biotechnological production for industrial applications

“…The biosensor-guided expression of ratelimiting enzymes may be used, as was shown for the tryptophan synthase β-subunit trpB gene which was expressed under the L-serine-responsive transcriptional activator SerR in the C. glutamicum cells for production of l-Trp derivatives [125]. The biotechnological production of indoles may be further accelerated in the next years due to advances in development of non-model microorganisms as microbial cell factories for the production of chemicals [126], acceleration of high-throughput strain construction [127], the automation of the cultivation platforms [128,129] and new cell cultivation approaches, such as the use of a segregostat in which in contrast to the chemostat cultivation, phenotypic diversification of microbial populations is better controlled [130]. Segregostat application is especially interesting in synthetic co-culture process of indole bioproduction since indole as previously described affects bacteria through signalling, i.e.…”

Automated in vivo enzyme engineering accelerates biocatalyst optimization

“…Most importantly, ML methods can be employed to enable autonomous exploration of the enzyme fitness landscape of combinatorial mutagenesis libraries 162 . In the same vein, reliable and autonomous growth phenotyping of resulting second-generation sensor strains has become possible by combining automated microbioreactor platforms 163 with appropriate data processing tools 164 .…”