CRISPR-mediated protein-tagging signal amplification systems for efficient transcriptional activation and repression in Saccharomyces cerevisiae.

Saccharomyces cerevisiae is an important model eukaryotic microorganism and widely applied in fundamental research and the production of various chemicals. Its ability to efficiently and precisely control the expression of multiple genes is valuable for metabolic engineering. The clustered regularly interspaced short palindromic repeats (CRISPR)-mediated regulation enables complex gene expression programming; however, the regulation efficiency is often limited by the efficiency of pertinent regulators. Here, we developed CRISPR-mediated protein-tagging signal amplification system for simultaneous multiplexed gene activation and repression in S. cerevisiae. By introducing protein scaffolds (SPY and SunTag systems) to recruit multiple copies of regulators to different nuclease-deficient CRISPR proteins and design optimization, our system amplified gene regulation efficiency significantly. The gene activation and repression efficiencies reached as high as 34.9-fold and 95%, respectively, being 3.8- and 8.6-fold higher than those observed on the direct fusion of regulators with nuclease-deficient CRISPR proteins, respectively. We then applied the orthogonal bifunctional CRISPR-mediated transcriptional regulation system to regulate the expression of genes associated with 3-hydroxypropanoic acid production to deduce that CRISPR-associated regulator recruiting systems represent a robust method for simultaneously regulating multiple genes and rewiring metabolic pathways.

[Recent advances of continuous in vivo evolution].

Laboratory evolution is an important approach to improve the performance of microorganisms. In the past decades, the methods for laboratory evolution have developed rapidly and applied widely. However, the commonly used evolution strategies for strains or specific proteins cannot achieve continuous mutation, and require multiple rounds of operation, therefore they are considered as a labor intensive process. The development of mutation and screening technologies have facilitated the development of continuous evolution in vivo and greatly improved the efficiency of laboratory evolution. The continuous in vivo evolution achieves in vivo mutation, perfectly combining mutation with screening to evolve a specific phenotype with minimal human intervention. This review summarizes the recent advances of in vivo continuous evolution technologies for either genome-scale mutation or evolution of specific proteins. The principles of these technologies and their applications are introduced. On this basis, the advantages and limitations of these technologies are discussed. We also give a perspective of future development of continuous in vivo evolution.

Evidence for rate-dependent filtering of global extrinsic noise by biochemical reactions in mammalian cells.

Recent studies have revealed that global extrinsic noise arising from stochasticity in the intracellular biochemical environment plays a critical role in heterogeneous cell physiologies. However, it remains largely unclear how such extrinsic noise dynamically influences downstream reactions and whether it could be neutralized by cellular reactions. Here, using fluorescent protein (FP) maturation as a model biochemical reaction, we explored how cellular reactions might combat global extrinsic noise in mammalian cells. We developed a novel single-cell assay to systematically quantify the maturation rate and the associated noise for over a dozen FPs. By exploiting the variation in the maturation rate for different FPs, we inferred that global extrinsic noise could be temporally filtered by maturation reactions, and as a result, the noise levels for slow-maturing FPs are lower compared to fast-maturing FPs. This mechanism is validated by directly perturbing the maturation rates of specific FPs and measuring the resulting noise levels. Together, our results revealed a potentially general principle governing extrinsic noise propagation, where timescale separation allows cellular reactions to cope with dynamic global extrinsic noise.

Biosensors design in yeast and applications in metabolic engineering.

Engineering microbial cell factories is a potential approach of sustainable production of chemicals, fuels and pharmaceuticals. However, testing the production of molecules in high throughput is still a time-consuming and laborious process since product synthesis usually does not confer a clear phenotype. Therefore, it is necessary to develop new techniques for fast high-producer screening. Genetically encoded biosensors are considered to be promising devices for high-throughput analysis owing to their ability to sense metabolites and couple detection to an actuator, thereby facilitating the rapid detection of small molecules at single-cell level. Here, we review recent advances in the design and engineering of biosensors in Saccharomyces cerevisiae, and their applications in metabolic engineering. Three types of biosensor are introduced in this review: transcription factor based, RNA-based and enzyme-coupled biosensors. The studies to improve the features of biosensors are also described. Moreover, we summarized their metabolic engineering applications in dynamic regulation and high producer selection. Current challenges in biosensor design and future perspectives on sensor applications are also discussed.

Developing synthetic hybrid promoters to increase constitutive or diauxic shift-induced expression in Saccharomyces cerevisiae.

Fine-tuning of the expression of genes is crucial for cell factory construction. Promoters are the most important tools to control gene expression. However, native promoters are often limited by their transcriptional ability. In this study, we sought to overcome the limitations of native promoters in Saccharomyces cerevisiae through the construction of hybrid promoter libraries for both constitutive promoters and promoters induced by diauxic shift. A series of hybrid constitutive promoters were constructed by combing the upstream activation sequences and changing the core promoter elements. The transcriptional capacity of the strongest promoter was 2-fold higher than that of the yeast native TEF1 promoter. Aside from the constitutive promoters, hybrid promoters that were induced in the post-diauxic phase were also constructed. These promoters had low transcriptional ability during growth on glucose and automatically activated upon growth with a diauxic shift. The strength of these promoters was also increased by replacing the core promoter with strong core promoters. Our study provides a series of constitutive and diauxic shift-induced promoters with a broad range of transcriptional capacity and will facilitate synthetic biology and metabolic engineering application.

The levels of oxidative stress and antioxidant capacity in hibernating Nanorana parkeri.

The effect of hibernation on oxidative stress and antioxidant defense was assessed in the frog Nanorana parkeri which inhabits the southern Tibetan Plateau. We compared the indices of oxidative stress (GSSG/GSH), the degree of oxidative damage (content of carbonyl proteins and lipid peroxide products) and the activities of antioxidant enzymes (SOD, CAT, GPx, GST and GR) in liver, brain, heart and muscle of N. parkeri sampled during summer and winter. Obtained results showed that hibernation induced a significant decrease in the level of GSH in heart, liver, and muscle, while the ratio of GSSG/GSH markedly increased in all tissues except for muscle. Regarding oxidative damage, significant increases in TBARS were observed in all tissues of N. parkeri in the midst of hibernation, and the lipid peroxides level also clearly elevated in these tissues except the liver. In liver and brain, the level of carbonyl proteins was significantly higher in winter relative to summer. Additionally, the activity of antioxidant enzymes obviously reduced in the liver of hibernating N. parkeri. The total antioxidant capacity was also significantly lower in all tissues during winter than summer. In conclusion, hibernation in N. parkeri induced oxidative stress which was supported by oxidative damage to lipids and proteins with suppression of antioxidant defense.