Towards robust Pseudomonas cell factories to harbour novel biosynthetic pathways

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.

Integrated rational and evolutionary engineering of genome-reducedPseudomonas putidastrains empowers synthetic formate assimilation

“…These results highlight the value of P . putida as a surrogate host of synthetic formatotrophy routes due to its high tolerance to stressful conditions (52)—in this case, brought about by the feedstock. Owing to its emergent phenotypic and metabolic properties [e.g.…”

Oligomerization engineering of the fluorinase enzyme leads to an active trimer that supports synthesis of fluorometabolites in vitro

“…Such building blocks are usually prepared by indirect fluorination techniques based on diazotization of anilines or exchange fluorination of activated haloaromatics and, more recently, by developing new fluorinating agents incorporated in somewhat complex synthetic methods (Cheng and Ritter, 2019 ). In addition, the emerging catalytic features of novel FlA variants can be also exploited in combination with robust microbial hosts (Volke et al ., 2020a ; Nikel et al ., 2021 ) that support the synthesis of fluorometabolites, compounds typically known to be toxic for the cells (Bitzenhofer et al ., 2021 ). Taken these results together, our present study highlights that the construction of bacterial cell factories for fluorochemicals production involving FlA will benefit from manipulating a variety of parameters – including the enzyme oligomerization status via direct, rational protein engineering – for optimal catalytic performance.…”

One Health Probiotics as Biocontrol Agents: One Health Tomato Probiotics

“…For example, bacterial metabolites may be a potential source of bioactive molecules, such as tripyrrolic, red-colored prodiginines, which not only have antimicrobial activity against plant pathogens but also have antinematodal activities [ 38 ]. The combined use of modern molecular genetic methods and toolsets, enzyme engineering, as well as biocatalysts and bioinformatics, make it possible to obtain many natural plant antimicrobial compounds [ 39 ]. Genome editing (GE) tools such as the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas-mediated GE have been recently used to study plant–microbe interactions for agronomic trait improvement [ 40 ].…”