Displaced by deceivers - Prevention of biosensor cross-talk is pivotal for successful biosensor-based high-throughput screening campaigns

Transcriptional biosensors emerged as powerful tools for protein and strain engineering as they link inconspicuous production phenotypes to easily measurable output signals such as fluorescence. When combined with fluorescence-activated cell sorting, transcriptional biosensors enable high throughput screening of vast mutant libraries. Interestingly, even though many published manuscripts describe the construction and characterization of transcriptional biosensors, only very few studies report the successful application of transcriptional biosensors in such high-throughput screening campaigns. Here, we describe construction and characterization of the trans-cinnamic acid responsive transcriptional biosensor pSenCA for Escherichia coli and its application in a FACS based screen. In this context, we focus on essential methodological challenges during the development of such biosensor-guided high-throughput screens such as biosensor cross-talk between producing and nonproducing cells, which could be minimized by optimization of expression and cultivation conditions. The optimized conditions were applied in a five-step FACS campaign and proved suitable to isolate phenylalanine ammonia lyase variants with improved activity in E. coli and in vitro. Findings from this study will help researchers who want to profit from the unmatched throughput of fluorescence-activated cell sorting by using transcriptional biosensors for their enzyme and strain engineering campaigns

Alone at last! – Heterologous expression of a single gene is sufficient for establishing the five-step Weimberg pathway in Corynebacterium glutamicum

Corynebacterium glutamicum can grow on d-xylose as sole carbon and energy source via the five-step Weimberg pathway when the pentacistronic xylXABCD operon from Caulobacter crescentus is heterologously expressed. More recently, it could be demonstrated that the C. glutamicum wild type accumulates the Weimberg pathway intermediate d-xylonate when cultivated in the presence of d-xylose. Reason for this is the activity of the endogenous dehydrogenase IolG, which can also oxidize d-xylose. This raised the question whether additional endogenous enzymes in C. glutamicum contribute to the catabolization of d-xylose via the Weimberg pathway. In this study, analysis of the C. glutamicum genome in combination with systematic reduction of the heterologous xylXABCD operon revealed that the hitherto unknown and endogenous dehydrogenase KsaD (Cg0535) can also oxidize α-ketoglutarate semialdehyde to the tricarboxylic acid cycle intermediate α-ketoglutarate, the final enzymatic step of the Weimberg pathway. Furthermore, heterologous expression of either xylX or xylD, encoding for the two dehydratases of the Weimberg pathway in C. crescentus, is sufficient for enabling C. glutamicum to grow on d-xylose as sole carbon and energy source. Finally, several variants for the carbon-efficient microbial production of α-ketoglutarate from d-xylose were constructed. In comparison to cultivation solely on d-glucose, the best strain accumulated up to 1.5-fold more α-ketoglutarate in d-xylose/d-glucose mixtures

Mutations in MurE, the essential UDP-N-acetylmuramoylalanyl-d-glutamate 2,6-diaminopimelate ligase of Corynebacterium glutamicum: effect on l-lysine formation and analysis of systemic consequences

ObjectivesTo explore systemic effects of mutations in the UDP-N-acetylmuramoylalanyl-d-glutamate 2,6-diaminopimelate ligase (MurE) of Corynebacterium glutamicum, that leads to extracellular l-lysine accumulation by this bacterium.ResultsThe analysis of a mutant cohort of C. glutamicum strains carrying all possible 20 amino acids at position 81 of MurE revealed unexpected effects on cellular properties. With increasing l-lysine accumulation the growth rate of the producing strain is reduced. A dynamic flux balance analysis including the flux over MurE fully supports this finding and suggests that further reductions at this flux control point would enhance l-lysine accumulation even further. The strain carrying the best MurE variant MurE-G81K produces 37 mM l-lysine with a yield of 0.17 g/g (l-lysine·HCl/glucose·H2O), bearing no other genetic modification. Interestingly, among the strains with high l-lysine titers, strain variants occur which, despite possessing the desired amino acid substitutions in MurE, have regained close to normal growth and correspondingly lower l-lysine accumulation. Genome analyses of such variants revealed the transposition of mobile genetic elements which apparently annulled the favorable consequences of the MurE mutations on l-lysine formation.ConclusionMurE is an attractive target to achieve high l-lysine accumulation, and product formation is inversely related to the specific growth rate. Moreover, single point mutations leading to elevated l-lysine titers may cause systemic effects on different levels comprising also major genome modifications. The latter caused by the activity of mobile genetic elements, most likely due to the stress conditions being characteristic for microbial metabolite producers

(Optochemical) Control of Synthetic Microbial Coculture Interactions on a Microcolony Level

Synthetic microbial cocultures carry enormous potential for applied biotechnology and are increasingly the subject of fundamental research. So far, most cocultures have been designed and characterized based on bulk cultivations without considering the potentially highly heterogeneous and diverse single-cell behavior. However, an in-depth understanding of cocultures including their interacting single cells is indispensable for the development of novel cultivation approaches and control of cocultures. We present the development, validation, and experimental characterization of an optochemically controllable bacterial coculture on a microcolony level consisting of two Corynebacterium glutamicum strains. Our coculture combines an l-lysine auxotrophic strain together with a l-lysine-producing variant carrying the genetically IPTG-mediated induction of l-lysine production. We implemented two control approaches utilizing IPTG as inducer molecule. First, unmodified IPTG was supplemented to the culture enabling a medium-based control of the production of l-lysine, which serves as the main interacting component. Second, optochemical control was successfully performed by utilizing photocaged IPTG activated by appropriate illumination. Both control strategies were validated studying cellular growth on a microcolony level. The novel microfluidic single-cell cultivation strategies applied in this work can serve as a blueprint to validate cellular control strategies of synthetic mono- and cocultures with single-cell resolution at defined environmental conditions

(Optochemical) Control of Synthetic Microbial Coculture Interactions on a Microcolony Level

Burmeister A, Akhtar Q, Hollmann L, et al. (Optochemical) Control of Synthetic Microbial Coculture Interactions on a Microcolony Level. ACS synthetic biology. 2021.Synthetic microbial cocultures carry enormous potential for applied biotechnology and are increasingly the subject of fundamental research. So far, most cocultures have been designed and characterized based on bulk cultivations without considering the potentially highly heterogeneous and diverse single-cell behavior. However, an in-depth understanding of cocultures including their interacting single cells is indispensable for the development of novel cultivation approaches and control of cocultures. We present the development, validation, and experimental characterization of an optochemically controllable bacterial coculture on a microcolony level consisting of two Corynebacterium glutamicum strains. Our coculture combines an l-lysine auxotrophic strain together with a l-lysine-producing variant carrying the genetically IPTG-mediated induction of l-lysine production. We implemented two control approaches utilizing IPTG as inducer molecule. First, unmodified IPTG was supplemented to the culture enabling a medium-based control of the production of l-lysine, which serves as the main interacting component. Second, optochemical control was successfully performed by utilizing photocaged IPTG activated by appropriate illumination. Both control strategies were validated studying cellular growth on a microcolony level. The novel microfluidic single-cell cultivation strategies applied in this work can serve as a blueprint to validate cellular control strategies of synthetic mono- and cocultures with single-cell resolution at defined environmental conditions