Comparative Single-Cell Analysis of Different E. coli Expression Systems during Microfluidic Cultivation

Binder D, Probst C, Grünberger A, et al. Comparative Single-Cell Analysis of Different E. coli Expression Systems during Microfluidic Cultivation. PLoS one. 2016;11(8): e0160711.Recombinant protein production is mostly realized with large-scale cultivations and monitored at the level of the entire population. Detailed knowledge of cell-to-cell variations with respect to cellular growth and product formation is limited, even though phenotypic heterogeneity may distinctly hamper overall production yields, especially for toxic or difficult-to-express proteins. Unraveling phenotypic heterogeneity is thus a key aspect in understanding and optimizing recombinant protein production in biotechnology and synthetic biology. Here, microfluidic single-cell analysis serves as the method of choice to investigate and unmask population heterogeneities in a dynamic and spatiotemporal fashion. In this study, we report on comparative microfluidic single-cell analyses of commonly used E. coli expression systems to uncover system-inherent specifications in the synthetic M9CA growth medium. To this end, the PT7lac/LacI, the PBAD/AraC and the Pm/XylS system were systematically analyzed in order to gain detailed insights into variations of growth behavior and expression phenotypes and thus to uncover individual strengths and deficiencies at the single-cell level. Specifically, we evaluated the impact of different system-specific inducers, inducer concentrations as well as genetic modifications that affect inducer-uptake and regulation of target gene expression on responsiveness and phenotypic heterogeneity. Interestingly, the most frequently applied expression system based on E. coli strain BL21(DE3) clearly fell behind with respect to expression homogeneity and robustness of growth. Moreover, both the choice of inducer and the presence of inducer uptake systems proved crucial for phenotypic heterogeneity. Conclusively, microfluidic evaluation of different inducible E. coli expression systems and setups identified the modified lacY-deficient PT7lac/LacI as well as the Pm/XylS system with conventional m-toluic acid induction as key players for precise and robust triggering of bacterial gene expression in E. coli in a homogeneous fashion

Light-responsive control of bacterial gene Expression: precise triggering of the lac promoter activity using photocaged IPTG

Binder D, Grünberger A, Loeschcke A, et al. Light-responsive control of bacterial gene Expression: precise triggering of the lac promoter activity using photocaged IPTG. Integrative biology. 2014;6(8):755-765.Light can be used to control numerous cellular processes including protein function and interaction as well as gene expression in a non-invasive fashion and with unprecedented spatiotemporal resolution. However, for chemical phototriggers tight, gradual, and homogeneous light response has never been attained in living cells. Here, we report on a light-responsive bacterial T7 RNA polymerase expression system based on a photocaged derivative of the inducer molecule isopropyl-β-D-thiogalactopyranoside (IPTG). We have comparatively analyzed different Escherichia coli lac promoter-regulated expression systems in batch and microfluidic single-cell cultivation. The lacY-deficient E. coli strain Tuner(DE3) harboring additional plasmid-born copies of the lacI gene exhibited a sensitive and defined response to increasing IPTG concentrations. Photocaged IPTG served as a synthetic photo-switch to convert the E. coli system into an optogenetic expression module allowing for precise and gradual light-triggering of gene expression as demonstrated at the single cell level. Graphical abstract: Light-responsive control of bacterial gene expression: precise triggering of the lac promoter activity using photocaged IPT

Engineering of natural product biosynthesis in Pseudomonas putida

Organisms from all kingdoms of life represent a rich source for valuable natural products with various applications. Microbial systems are useful to implement the expression of respective biosynthetic genes and establish the production and effective access to natural products, or to elucidate the biochemistry of the underlying pathways. Pseudomonas putida, a Gram-negative soil bacterium, appears to be particularly suitable for natural product biosynthesis because of an advantageous intrinsic metabolism and a remarkable tolerance toward various xenobiotics. This article presents the current state of engineering P. putida for the biosynthesis of rhamnolipids, terpenoids, polyketides, non-ribosomal peptides, and products from amino acid metabolism. Technological advances facilitating chromosomal integration and expression of biosynthetic genes, and smart metabolic engineering are key to success

Pseudomonas putida-a versatile host for the production of natural products

The biosynthesis of natural products by heterologous expression of biosynthetic pathways in amenable production strains enables biotechnological access to a variety of valuable compounds by conversion of renewable resources. Pseudomonas putida has emerged as a microbial laboratory work horse, with elaborated techniques for cultivation and genetic manipulation available. Beyond that, this bacterium offers several particular advantages with regard to natural product biosynthesis, notably a versatile intrinsic metabolism with diverse enzymatic capacities as well as an outstanding tolerance to xenobiotics. Therefore, it has been applied for recombinant biosynthesis of several valuable natural products. This review provides an overview of applications of P. putida as a host organism for the recombinant biosynthesis of such natural products, including rhamnolipids, terpenoids, polyketides and non-ribosomal peptides, and other amino acid-derived compounds. The focus is on de novo natural product synthesis from intrinsic building blocks by means of heterologous gene expression and strain engineering. Finally, the future potential of the bacterium as a chassis organism for synthetic microbiology is pointed out