Towards robust Pseudomonas cell factories to harbour novel biosynthetic pathways

Ekaterina Kozaeva,Thorsten Lechtenberg,Luzie Kruse,Karl-Erich Jaeger,Pablo Iván Nikel,Stephan Thies,Nick Wierckx,Jakob Rönitz,Anita Loeschcke,Hermann J Heipieper,Nicolas Thilo Wirth,Nora Lisa Bitzenhofer,Christian Eberlein,Benedikt Wynands,Diethard Mattanovich,Pablo Ivan Nikel

doi:10.1042/ebc20200173

Abstract

Biotechnological production in bacteria enables access to numerous valuable chemical compounds. Nowadays, advanced molecular genetic toolsets, enzyme engineering as well as the combinatorial use of biocatalysts, pathways, and circuits even bring new-to-nature compounds within reach. However, the associated substrates and biosynthetic products often cause severe chemical stress to the bacterial hosts. Species of the Pseudomonas clade thus represent especially valuable chassis as they are endowed with multiple stress response mechanisms, which allow them to cope with a variety of harmful chemicals. A built-in cell envelope stress response enables fast adaptations that sustain membrane integrity under adverse conditions. Further, effective export machineries can prevent intracellular accumulation of diverse harmful compounds. Finally, toxic chemicals such as reactive aldehydes can be eliminated by oxidation and stress-induced damage can be recovered. Exploiting and engineering these features will be essential to support an effective production of natural compounds and new chemicals. In this article, we therefore discuss major resistance strategies of Pseudomonads along with approaches pursued for their targeted exploitation and engineering in a biotechnological context. We further highlight strategies for the identification of yet unknown tolerance-associated genes and their utilisation for engineering next-generation chassis and finally discuss effective measures for pathway fine-tuning to establish stable cell factories for the effective production of natural compounds and novel biochemicals.

Highlights

Microbial biotechnology can provide chemical compounds that are essential for modern societies in multiple sectors, e.g. as pharmaceuticals and chemical building blocks
• Efficient bioproduction of chemical compounds using whole cells as biocatalysts is often hampered by the limited ability of microbial hosts to cope with the stress linked to production
• Pseudomonads are endowed with effective stress responses to many chemicals

Summary

Introduction

Microbial biotechnology can provide chemical compounds that are essential for modern societies in multiple sectors, e.g. as pharmaceuticals and chemical building blocks. Bacteria have evolved numerous strategies to alleviate chemical stress and members of the Pseudomonas clade are especially well-equipped with such traits [1] This has likely contributed to the development of the soil bacterium Pseudomonas putida and its relatives into versatile microbial cell factories during the past few decades, enabling the biosynthesis of various compounds including secondary metabolites like rhamnolipids, terpenes, polyketides, and non-ribosomal peptides, organic acids, alcohols, and aromatics [2,3,4]. The genetic basis of OMV formation is not yet entirely understood but could be connected to a range of genes [37] Such knowledge has already been successfully applied for the engineering of hypervesiculation to support cell viability under stress conditions [46] or protein secretion in E. coli [47], but has not yet been utilised in the context of optimising Pseudomonas biotechnology and will certainly require fine-tuning to avoid extensive loss of lipids and membrane integrity [48,49]. The above-mentioned urgent response mechanisms enable Pseudomonas species to react quickly to emerging adverse conditions, making them robust candidates for whole-cell biotransformation processes and accessing new-to-nature chemistry

Aim

Conclusion

Summary