Optimization of a whole-cell biocatalyst by employing genetically encoded product sensors inside nanolitre reactors

Meyer, Andreas; Pellaux, René; Potot, Sébastien; Becker, Katja; Hohmann, Hans‐Peter; Panke, Sven; Held, Martin

doi:10.1038/nchem.2301

Cited by 95 publications

(70 citation statements)

References 34 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Metabolic engineering by means of recombinant DNA technology result in a large number of genetic variants to be tested. According to Meyer et al ., most laboratories working on metabolic engineering at the millilitre scale can easily generate libraries with up to 19 9 genetically different isolates (Meyer et al ., ). Since in industrial settings, high‐throughput screening assays include up to 10 4 variants per evolution round, the authors proposed a method based on microcompartmentalization.…”

Section: Improving Microorganismsmentioning

confidence: 97%

Whole cell biocatalysts: essential workers from Nature to the industry

Carvalho

2016

Microbial Biotechnology

213

157

View full text Add to dashboard Cite

SummaryMicroorganisms have been exposed to a myriad of substrates and environmental conditions throughout evolution resulting in countless metabolites and enzymatic activities. Although mankind have been using these properties for centuries, we have only recently learned to control their production, to develop new biocatalysts with high stability and productivity and to improve their yields under new operational conditions. However, microbial cells still provide the best known environment for enzymes, preventing conformational changes in the protein structure in non‐conventional medium and under harsh reaction conditions, while being able to efficiently regenerate necessary cofactors and to carry out cascades of reactions. Besides, a still unknown microbe is probably already producing a compound that will cure cancer, Alzeihmer's disease or kill the most resistant pathogen. In this review, the latest developments in screening desirable activities and improving production yields are discussed.

show abstract

Section: Improving Microorganismsmentioning

confidence: 97%

Whole cell biocatalysts: essential workers from Nature to the industry

Carvalho

2016

Microbial Biotechnology

213

157

View full text Add to dashboard Cite

show abstract

“…In this study, we took advantage of the natural fluorescence of riboflavin for detection, but other approaches can easily be used as well. The recent development of GFP-based biosensor technology has been successfully applied to isolate microorganisms producing riboflavin and other compounds (9, 30). …”

Section: Discussionmentioning

confidence: 99%

“…The small size of the droplets, whose volumes are measured in nanoliters or picoliters, allows affordable HTS with much reduced costs for consumables (8). DBM has been used in several screening applications, such as optimization of cell factories for the production of both recombinant proteins and chemicals (6, 7, 9–11). Some studies have also applied this technique to screen libraries generated using whole-genome random mutagenesis (10, 11).…”

Section: Introductionmentioning

confidence: 99%

Finding the Needle in the Haystack—the Use of Microfluidic Droplet Technology to Identify Vitamin-Secreting Lactic Acid Bacteria

Chen

Vestergaard

Jensen

et al. 2017

mBio

View full text Add to dashboard Cite

Efficient screening technologies aim to reduce both the time and the cost required for identifying rare mutants possessing a phenotype of interest in a mutagenized population. In this study, we combined a mild mutagenesis strategy with high-throughput screening based on microfluidic droplet technology to identify Lactococcus lactis variants secreting vitamin B 2 (riboflavin). Initially, we used a roseoflavinresistant mutant of L. lactis strain MG1363, JC017, which secreted low levels of riboflavin. By using fluorescence-activated droplet sorting, several mutants that secreted riboflavin more efficiently than JC017 were readily isolated from the mutagenesis library. The screening was highly efficient, and candidates with as few as 1.6 mutations per million base pairs (Mbp) were isolated. The genetic characterization revealed that riboflavin production was triggered by mutations inhibiting purine biosynthesis, which is surprising since the purine nucleotide GTP is a riboflavin precursor. Purine starvation in the mutants induced overexpression of the riboflavin biosynthesis cluster ribABGH. When the purine starvation was relieved by purine supplementation in the growth medium, the outcome was an immediate downregulation of the riboflavin biosynthesis cluster and a reduction in riboflavin production. Finally, by applying the new isolates in milk fermentation, the riboflavin content of milk (0.99 mg/liter) was improved to 2.81 mg/liter, compared with 0.66 mg/liter and 1.51 mg/liter by using the wild-type strain and the original roseoflavin-resistant mutant JC017, respectively. The results obtained demonstrate how powerful classical mutagenesis can be when combined with droplet-based microfluidic screening technology for obtaining microorganisms with useful attributes. IMPORTANCEThe food industry prefers to use classical approaches, e.g., random mutagenesis followed by screening, to improve microorganisms used in food production, as the use of recombinant DNA technologies is still not widely accepted. Although modern automated screening platforms are widely accessible, screening remains as a bottleneck in strain development, especially when a mild mutagenesis approach is applied to reduce the chance of accumulating unintended mutations, which may cause unwanted phenotypic changes. Here, we incorporate a droplet-based high-throughput screening method into the strain development process and readily capture L. lactis variants with more efficient vitamin secretion from low-error-rate mutagenesis libraries. This study shows that useful mutants showing strong phenotypes but without extensive mutations can be identified with efficient screening technologies. It is therefore possible to avoid accumulating detrimental mutations while enriching beneficial ones through iterative mutagenesis screening. Due to the low mutation rates, the genetic determinants are also readily identified.

show abstract

“…Whole cell biosensors can be created without the need for complicated protein or aptamer engineering 144 . For example, recombinant E. coli made auxotrophic for mevalonate shows concentration-dependent growth rate changes in the presence of extracellular mevalonate 145 .…”

Section: Genetically-encoded Biosensors For Biosynthetic Pathway Engimentioning

confidence: 99%

Synthetic biology to access and expand nature's chemical diversity

et al. 2016

View full text Add to dashboard Cite

Bacterial genomes encode the biosynthetic potential to produce hundreds of thousands of complex molecules with diverse applications, from medicine to agriculture and materials. Economically accessing the potential encoded within sequenced genomes promises to reinvigorate waning drug discovery pipelines and provide novel routes to intricate chemicals. This is a tremendous undertaking, as the pathways often comprise dozens of genes spanning as much as 100+ kiliobases of DNA, are controlled by complex regulatory networks, and the most interesting molecules are made by non-model organisms. Advances in synthetic biology address these issues, including DNA construction technologies, genetic parts for precision expression control, synthetic regulatory circuits, computer aided design, and multiplexed genome engineering. Collectively, these technologies are moving towards an era when chemicals can be accessed en mass based on sequence information alone. This will enable the harnessing of metagenomic data and massive strain banks for high-throughput molecular discovery and, ultimately, the ability to forward design pathways to complex chemicals not found in nature.

show abstract

Optimization of a whole-cell biocatalyst by employing genetically encoded product sensors inside nanolitre reactors

Cited by 95 publications

References 34 publications

Whole cell biocatalysts: essential workers from Nature to the industry

Whole cell biocatalysts: essential workers from Nature to the industry

Finding the Needle in the Haystack—the Use of Microfluidic Droplet Technology to Identify Vitamin-Secreting Lactic Acid Bacteria

Synthetic biology to access and expand nature's chemical diversity

Contact Info

Product

Resources

About