Rational design of a bacterial import system for new-to-nature molecules.

Integration of novel compounds into biological processes holds significant potential for modifying or expanding existing cellular functions. However, the cellular uptake of these compounds is often hindered by selectively permeable membranes. We present a novel bacterial transport system that has been rationally designed to address this challenge. Our approach utilizes a highly promiscuous sulfonate membrane transporter, which allows the passage of cargo molecules attached as amides to a sulfobutanoate transport vector molecule into the cytoplasm of the cell. These cargoes can then be unloaded from the sulfobutanoyl amides using an engineered variant of the enzyme γ-glutamyl transferase, which hydrolyzes the amide bond and releases the cargo molecule within the cell. Here, we provide evidence for the broad substrate specificity of both components of the system by evaluating a panel of structurally diverse sulfobutanoyl amides. Furthermore, we successfully implement the synthetic uptake system in vivo and showcase its functionality by importing an impermeant non-canonical amino acid.

Plasmid replication based on the T7 origin of replication requires a T7 RNAP variant and inactivation of ribonuclease H.

T7 RNA polymerase (RNAP) is a valuable tool in biotechnology, basic research and synthetic biology due to its robust, efficient and selective transcription of genes. Here, we expand the scope of T7 RNAP to include plasmid replication. We present a novel type of plasmid, termed T7 ori plasmids that replicate, in an engineered Escherichia coli, with a T7 phage origin as the sole origin of replication. We find that while the T7 replication proteins; T7 DNA polymerase, T7 single-stranded binding proteins and T7 helicase-primase are dispensable for replication, T7 RNAP is required, although dependent on a T7 RNAP variant with reduced activity. We also find that T7 RNAP-dependent replication of T7 ori plasmids requires the inactivation of cellular ribonuclease H. We show that the system is portable among different plasmid architectures and ribonuclease H-inactivated E. coli strains. Finally, we find that the copy number of T7 ori plasmids can be tuned based on the induction level of RNAP. Altogether, this study assists in the choice of an optimal genetic tool by providing a novel plasmid that requires T7 RNAP for replication.

Development and optimisation of a defined high cell density yeast medium.

Saccharomyces cerevisiae cells grown in a small volume of chemically defined media neither reach the desired cell density nor grow at a fast enough rate to scale down the volume and increase the sample number of classical biochemical assays, as the detection limit of the readout often requires a high number of cells as an input. To ameliorate this problem, we developed and optimised a new high cell density (HCD) medium for S. cerevisiae. Starting from a widely used synthetic medium composition, we systematically varied the concentrations of all components without the addition of other compounds. We used response surface methodology to develop and optimise the five components of the medium: glucose, yeast nitrogen base, amino acids, monosodium glutamate, and inositol. We monitored growth, cell number, and cell size to ensure that the optimisation was towards a greater density of cells rather than just towards an increase in biomass (i.e., larger cells). Cells grown in the final medium, HCD, exhibit growth more similar to the complex medium yeast extract peptone dextrose (YPD) than to the synthetic defined (SD) medium. Whereas the final cell density of HCD prior to the diauxic shift is increased compared with YPD and SD about threefold and tenfold, respectively. We found normal cell-cycle behaviour throughout the growth phases by monitoring DNA content and protein expression using fluorescent reporters. We also ensured that HCD media could be used with a variety of strains and that they allow selection for all common yeast auxotrophic markers.

Sequence-based prediction of permissive stretches for internal protein tagging and knockdown.

BACKGROUND: Internal tagging of proteins by inserting small functional peptides into surface accessible permissive sites has proven to be an indispensable tool for basic and applied science. Permissive sites are typically identified by transposon mutagenesis on a case-by-case basis, limiting scalability and their exploitation as a system-wide protein engineering tool. METHODS: We developed an apporach for predicting permissive stretches (PSs) in proteins based on the identification of length-variable regions (regions containing indels) in homologous proteins. RESULTS: We verify that a protein's primary structure information alone is sufficient to identify PSs. Identified PSs are predicted to be predominantly surface accessible; hence, the position of inserted peptides is likely suitable for diverse applications. We demonstrate the viability of this approach by inserting a Tobacco etch virus protease recognition site (TEV-tag) into several PSs in a wide range of proteins, from small monomeric enzymes (adenylate kinase) to large multi-subunit molecular machines (ATP synthase) and verify their functionality after insertion. We apply this method to engineer conditional protein knockdowns directly in the Escherichia coli chromosome and generate a cell-free platform with enhanced nucleotide stability. CONCLUSIONS: Functional internally tagged proteins can be rationally designed and directly chromosomally implemented. Critical for the successful design of protein knockdowns was the incorporation of surface accessibility and secondary structure predictions, as well as the design of an improved TEV-tag that enables efficient hydrolysis when inserted into the middle of a protein. This versatile and portable approach can likely be adapted for other applications, and broadly adopted. We provide guidelines for the design of internally tagged proteins in order to empower scientists with little or no protein engineering expertise to internally tag their target proteins.

Exploiting racemases.

Chiral resolutions of racemic mixtures are limited to a theoretical yield of 50 %. This yield can be doubled by integration of a step-wise or continuous racemization of the non-desired enantiomer. Many of the different routes along which the racemization step can be conducted require harsh treatments and are therefore often incompatible with the highly functionalized state of many compounds relevant for the life science industries. Employing enzymatic catalysis for racemization can therefore be highly beneficial. Racemases allow racemization in one reaction step. Most representatives from this group are found in the domain of amino acid or amino acid derivative racemization, with few other examples, notably the racemization of mandelic acid. Corresponding to the importance of enantiospecific conversion of amino acid precursor racemates for the production of enantiopure amino acids, the most important biotechnological use for racemases is the racemization of such precursors. However, alternative uses, in particular for mandelate and amino acid racemases, are emerging. Here, we summarize the natural roles of racemases and their occurrence, the applications, and the biochemistry and engineering of this promising class of biocatalysts.

Characterizing and Tailoring the Substrate Profile of a γ-Glutamyltransferase Variant.

Xenobiology is an emerging field that focuses on the extension and redesign of biological systems through the use of laboratory-derived xenomolecules, which are molecules that are new to the metabolism of the cell. Despite the enormous potential of using xenomolecules in living organisms, most noncanonical building blocks still need to be supplied externally, and often poor uptake into cells limits wider applicability. To improve the cytosolic availability of noncanonical molecules, a synthetic transport system based on portage transport was developed, in which molecules of interest "cargo" are linked to a synthetic transport vector that enables piggyback transport through the alkylsulfonate transporter (SsuABC) of Escherichia coli. Upon cytosolic delivery, the vector-cargo conjugate is enzymatically cleaved by GGTxe, leading to the release of the cargo molecule. To deepen our understanding of the synthetic transport system, we focused on the characterization and further development of the enzymatic cargo release step. Hence, the substrate scope of GGTxe was characterized using a library of structurally diverse vector-cargo conjugates and MS/MS-based quantification of hydrolysis products in a kinetic manner. The resulting substrate tolerance characterization revealed that vector-amino acid conjugates were significantly unfavored. To overcome this shortcoming, a selection system based on metabolic auxotrophy complementation and directed evolution of GGTxe was established. In a directed evolution campaign, we improved the enzymatic activity of GGTxe for vector-amino acid conjugates and revealed the importance of residue D386 in the cargo unloading step.

Standardization of Synthetic Biology Tools and Assembly Methods for Saccharomyces cerevisiae and Emerging Yeast Species.

As redesigning organisms using engineering principles is one of the purposes of synthetic biology (SynBio), the standardization of experimental methods and DNA parts is becoming increasingly a necessity. The synthetic biology community focusing on the engineering of Saccharomyces cerevisiae has been in the foreground in this area, conceiving several well-characterized SynBio toolkits widely adopted by the community. In this review, the molecular methods and toolkits developed for S. cerevisiae are discussed in terms of their contributions to the required standardization efforts. In addition, the toolkits designed for emerging nonconventional yeast species including Yarrowia lipolytica, Komagataella phaffii, and Kluyveromyces marxianus are also reviewed. Without a doubt, the characterized DNA parts combined with the standardized assembly strategies highlighted in these toolkits have greatly contributed to the rapid development of many metabolic engineering and diagnostics applications among others. Despite the growing capacity in deploying synthetic biology for common yeast genome engineering works, the yeast community has a long journey to go to exploit it in more sophisticated and delicate applications like bioautomation.

Corrigendum: Identification and Characterisation of a pH-stable GFP.

This corrects the article DOI: 10.1038/srep28166.

Efficient engineering of chromosomal ribosome binding site libraries in mismatch repair proficient Escherichia coli.

Multiplexed gene expression optimization via modulation of gene translation efficiency through ribosome binding site (RBS) engineering is a valuable approach for optimizing artificial properties in bacteria, ranging from genetic circuits to production pathways. Established algorithms design smart RBS-libraries based on a single partially-degenerate sequence that efficiently samples the entire space of translation initiation rates. However, the sequence space that is accessible when integrating the library by CRISPR/Cas9-based genome editing is severely restricted by DNA mismatch repair (MMR) systems. MMR efficiency depends on the type and length of the mismatch and thus effectively removes potential library members from the pool. Rather than working in MMR-deficient strains, which accumulate off-target mutations, or depending on temporary MMR inactivation, which requires additional steps, we eliminate this limitation by developing a pre-selection rule of genome-library-optimized-sequences (GLOS) that enables introducing large functional diversity into MMR-proficient strains with sequences that are no longer subject to MMR-processing. We implement several GLOS-libraries in Escherichia coli and show that GLOS-libraries indeed retain diversity during genome editing and that such libraries can be used in complex genome editing operations such as concomitant deletions. We argue that this approach allows for stable and efficient fine tuning of chromosomal functions with minimal effort.

Identification and Characterisation of a pH-stable GFP.

Green fluorescent proteins (GFPs) are invaluable tools for modern cell biology. Even though many properties of GFP have been successfully engineered, a GFP retaining brightness at low pH has not emerged. This limits the use of GFP in quantitative studies performed in fluctuating or acidic conditions. We report the engineering and characterisation of tandem dimer GFP (pH-tdGFP), a bright and stable GFP that can be efficiently excited and maintains its fluorescence properties in acidic conditions. Therefore, pH-tdGFP could act as a quantitative marker for cellular processes that occur at low pH, such as endocytosis, autophagy or starvation.