The Ongoing Quest to Crack the Genetic Code for Protein Production.

Understanding the genetic design principles that determine protein production remains a major challenge. Although the key principles of gene expression were discovered 50 years ago, additional factors are still being uncovered. Both protein-coding and non-coding sequences harbor elements that collectively influence the efficiency of protein production by modulating transcription, mRNA decay, and translation. The influences of many contributing elements are intertwined, which complicates a full understanding of the individual factors. In natural genes, a functional balance between these factors has been obtained in the course of evolution, whereas for genetic-engineering projects, our incomplete understanding still limits optimal design of synthetic genes. However, notable advances have recently been made, supported by high-throughput analysis of synthetic gene libraries as well as by state-of-the-art biomolecular techniques. We discuss here how these advances further strengthen understanding of the gene expression process and how they can be harnessed to optimize protein production.

Replacing the Calvin cycle with the reductive glycine pathway in Cupriavidus necator.

Formate can be directly produced from CO2 and renewable electricity, making it a promising microbial feedstock for sustainable bioproduction. Cupriavidus necator is one of the few biotechnologically-relevant hosts that can grow on formate, but it uses the Calvin cycle, the high ATP cost of which limits biomass and product yields. Here, we redesign C. necator metabolism for formate assimilation via the synthetic, highly ATP-efficient reductive glycine pathway. First, we demonstrate that the upper pathway segment supports glycine biosynthesis from formate. Next, we explore the endogenous route for glycine assimilation and discover a wasteful oxidation-dependent pathway. By integrating glycine biosynthesis and assimilation we are able to replace C. necator's Calvin cycle with the synthetic pathway and achieve formatotrophic growth. We then engineer more efficient glycine metabolism and use short-term evolution to optimize pathway activity. The final growth yield we achieve (2.6 gCDW/mole-formate) nearly matches that of the WT strain using the Calvin Cycle (2.9 gCDW/mole-formate). We expect that further rational and evolutionary optimization will result in a superior formatotrophic C. necator strain, paving the way towards realizing the formate bio-economy.

CRISPR with a Happy Ending: Non-Templated DNA Repair for Prokaryotic Genome Engineering.

The exploration of microbial metabolism is expected to support the development of a sustainable economy and tackle several problems related to the burdens of human consumption. Microorganisms have the potential to catalyze processes that are currently unavailable, unsustainable and/or inefficient. Their metabolism can be optimized and further expanded using tools like the clustered regularly interspaced short palindromic repeats and their associated proteins (CRISPR-Cas) systems. These tools have revolutionized the field of biotechnology, as they greatly streamline the genetic engineering of organisms from all domains of life. CRISPR-Cas and other nucleases mediate double-strand DNA breaks, which must be repaired to prevent cell death. In prokaryotes, these breaks can be repaired through either homologous recombination, when a DNA repair template is available, or through template-independent end joining, of which two major pathways are known. These end joining pathways depend on different sets of proteins and mediate DNA repair with different outcomes. Understanding these DNA repair pathways can be advantageous to steer the results of genome engineering experiments. In this review, we discuss different strategies for the genetic engineering of prokaryotes through either non-homologous end joining (NHEJ) or alternative end joining (AEJ), both of which are independent of exogenous DNA repair templates.

Bicistronic Design-Based Continuous and High-Level Membrane Protein Production in Escherichia coli.

Escherichia coli has been widely used as a platform microorganism for both membrane protein production and cell factory engineering. The current methods to produce membrane proteins in this organism require the induction of target gene expression and often result in unstable, low yields. Here, we present a method combining a constitutive promoter with a library of bicistronic design (BCD) elements, which enables inducer-free, tuned translation initiation for optimal protein production. Our system mediates stable, constitutive production of bacterial membrane proteins at yields that outperform those obtained with E. coli Lemo21(DE3), the current gold standard for bacterial membrane protein production. We envisage that the continuous, fine-tunable, and high-level production of membrane proteins by our method will greatly facilitate their study and their utilization in engineering cell factories.

Characterizing a thermostable Cas9 for bacterial genome editing and silencing.

CRISPR-Cas9-based genome engineering tools have revolutionized fundamental research and biotechnological exploitation of both eukaryotes and prokaryotes. However, the mesophilic nature of the established Cas9 systems does not allow for applications that require enhanced stability, including engineering at elevated temperatures. Here we identify and characterize ThermoCas9 from the thermophilic bacterium Geobacillus thermodenitrificans T12. We show that in vitro ThermoCas9 is active between 20 and 70 °C, has stringent PAM-preference at lower temperatures, tolerates fewer spacer-protospacer mismatches than SpCas9 and its activity at elevated temperatures depends on the sgRNA-structure. We develop ThermoCas9-based engineering tools for gene deletion and transcriptional silencing at 55 °C in Bacillus smithii and for gene deletion at 37 °C in Pseudomonas putida. Altogether, our findings provide fundamental insights into a thermophilic CRISPR-Cas family member and establish a Cas9-based bacterial genome editing and silencing tool with a broad temperature range.