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Background The soil bacterium Pseudomonas putida KT2440 is a “generally recognized as safe”-certified strain with robust property and versatile metabolism. Thus, it is an ideal candidate for synthetic biology, biodegradation, and other biotechnology applications. The known genome editing approaches of Pseudomonas are suboptimal; thus, it is necessary to develop a high efficiency genome editing tool. Results In this study, we established a fast and convenient CRISPR–Cas9 method in P. putida KT2440. Gene deletion, gene insertion and gene replacement could be achieved within 5 days, and the mutation efficiency reached > 70%. Single nucleotide replacement could be realized, overcoming the limitations of protospacer adjacent motif sequences. We also applied nuclease-deficient Cas9 binding at three locations upstream of enhanced green fluorescent protein (eGFP) for transcriptional inhibition, and the expression intensity of eGFP reduced to 28.5, 29.4, and 72.1% of the control level, respectively. Furthermore, based on this CRISPR–Cas9 system, we also constructed a CRISPR–Cpf1 system, which we validated for genome editing in P. putida KT2440. Conclusions In this research, we established CRISPR based genome editing and regulation control systems in P. putida KT2440. These fast and efficient approaches will greatly facilitate the application of P. putida KT2440.

Background Rational metabolic pathway engineering is capable of boosting upstream flux towards downstream synthesis of target products, such as aromatic amino acid derivatives. However, coordinated synthesis of multiple downstream derivatives faces difficulty of combinatorial optimization of cellular metabolism. Results We developed a strategy combining metabolic engineering optimization with the global transcriptional regulation of transcription factors (TFs) Spt15p and Gcn4p to optimize the synthesis of aromatic amino acid derivatives in yeast. It is verified that the special mutants of these TFs can respectively improve the biosynthesis of betaxanthin, a tyrosine derived edible pigment. Comparative transcriptome analysis shows that significant transcriptional tuning occurs in glycolysis, pentose phosphate pathway, aromatic amino acid synthesis pathways, etc. In addition, global transcriptional engineering is proved to enhance the coordinated biosynthesis of both tyrosine derived pigment betaxanthin and tryptophan derived pigment violacein by more than 50%. Finally, we obtain an optimized production of 208 mg/L betaxanthin in yeast cells by flask fermentation. Conclusions Our strategy supplies an effective way to optimize the coordinated synthesis of two structurally divergent pigments downstream of the common aromatic amino acid pathway.

Background Classical strain engineering methods often have limitations in altering multigenetic cellular phenotypes. Here we try to improve Saccharomyces cerevisiae ethanol tolerance and productivity by reprogramming its transcription profile through rewiring its key transcription component RNA polymerase II (RNAP II), which plays a central role in synthesizing mRNAs. This is the first report on using directed evolution method to engineer RNAP II to alter S. cerevisiae strain phenotypes. Results Error-prone PCR was employed to engineer the subunit Rpb7 of RNAP II to improve yeast ethanol tolerance and production. Based on previous studies and the presumption that improved ethanol resistance would lead to enhanced ethanol production, we first isolated variant M1 with much improved resistance towards 8 and 10% ethanol. The ethanol titers of M1 was ~122 g/L (96.58% of the theoretical yield) under laboratory very high gravity (VHG) fermentation, 40% increase as compared to the control. DNA microarray assay showed that 369 genes had differential expression in M1 after 12 h VHG fermentation, which are involved in glycolysis, alcoholic fermentation, oxidative stress response, etc. Conclusions This is the first study to demonstrate the possibility of engineering eukaryotic RNAP to alter global transcription profile and improve strain phenotypes. Targeting subunit Rpb7 of RNAP II was able to bring differential expression in hundreds of genes in S. cerevisiae, which finally led to improvement in yeast ethanol tolerance and production.

Microbes have long been used in the industry to produce valuable biochemicals. Combinatorial engineering approaches, new strain engineering tools derived from inverse metabolic engineering, have started to attract attention in recent years, including genome shuffling, error-prone DNA polymerase, global transcription machinery engineering (gTME), random knockout/overexpression libraries, ribosome engineering, multiplex automated genome engineering (MAGE), customized optimization of metabolic pathways by combinatorial transcriptional engineering (COMPACTER), and library construction of “tunable intergenic regions” (TIGR). Since combinatorial approaches and high-throughput screening methods are fundamentally interconnected, color/fluorescence-based, growth-based, and biosensor-based high-throughput screening methods have been reviewed. We believe that with the help of metabolic engineering tools and new combinatorial approaches, plus effective high-throughput screening methods, researchers will be able to achieve better results on improving microorganism performance under stress or enhancing biochemical yield.

Cyclic AMP receptor protein (CRP) is one of the seven global regulators in Escherichia coli, which regulates the expression of over 490 genes. It contains a cAMP binding N-terminal domain and a DNA binding C-terminal domain, connected via a short hinge region. Various stress-tolerant E. coli mutants had been obtained through transcriptional engineering of CRP. This review aims to shed some light on the possible mechanism behind these CRP variants, from the change in CRP structure, transcription profile, and DNA binding affinity. The amino acid substitutions are distributed along the protein—certain mutations have shown higher frequency than others, such as T127N and D138Y. β-Galactosidase reporter gene assay revealed that CRP mutants had lower binding affinity with all three classes of CRP-dependent promoters as compared to native CRP, which probably would change cellular transcription profile. Different CRP mutants would induce different cellular transcription profile in E. coli, but there are common genes differentially expressed in these variants, including upregulated gadAB and downregulated nontransporter genes aspA and tnaA, and transporter/poringenes malE, mglB, cstA, and lamB. We believe that transcriptional engineering of CRP can provide an alternative strain engineering method for E. coli and its detailed mechanism may need further investigations.

Cyanobacteria can function as solar-driven biofactories thanks to their ability to perform photosynthesis and the ease with which they are genetically modified. In this review, we discuss transcriptional parts and promoters available for engineering cyanobacteria. First, we go through special cyanobacterial characteristics that may impact engineering, including the unusual cyanobacterial RNA polymerase, sigma factors and promoter types, mRNA stability, circadian rhythm, and gene dosage effects. Then, we continue with discussing component characteristics that are desirable for synthetic biology approaches, including decoupling, modularity, and orthogonality. We then summarize and discuss the latest promoters for use in cyanobacteria regarding characteristics such as regulation, strength, and dynamic range and suggest potential uses. Finally, we provide an outlook and suggest future developments that would advance the field and accelerate the use of cyanobacteria for renewable biotechnology.

One major challenge in biofuel production, including biobutanol production, is the low tolerance of the microbial host towards increasing biofuel concentration during fermentation. Here, we have demonstrated that Escherichia coli 1-butanol tolerance can be greatly enhanced through random mutagenesis of global transcription factor cyclic AMP receptor protein (CRP). Four mutants (MT1-MT4) with elevated 1-butanol tolerance were isolated from error-prone PCR libraries through an enrichment screening. A DNA shuffling library was then constructed using MT1-MT4 as templates and one mutant (MT5) that exhibited the best tolerance ability among all variants was selected. In the presence of 0.8 % (v/v, 6.5 g/l) 1-butanol, the growth rate of MT5 was found to be 0.28 h^sup -1^ while that of wild type was 0.20 h^sup -1^. When 1-butanol concentration increased to 1.2 % (9.7 g/l), the growth rate of MT5 (0.18 h^sup -1^) became twice that of the wild type (0.09 h^sup -1^). Microbial adhesion to hydrocarbon test showed that cell surface of MT5 was less hydrophobic and its cell length became significantly longer in the presence of 1-butanol, as observed by scanning electron microscopy. Quantitative real-time reverse transcription PCR analysis revealed that several CRP regulated, 1-butanol stress response related genes (rpoH, ompF, sodA, manX, male, and marA) demonstrated differential expression in MT5 in the presence or absence of 1-butanol. In conclusion, direct manipulation of the transcript profile through engineering global transcription factor CRP can provide a useful tool in strain engineering. [PUBLICATION ABSTRACT]

Industrial strains have been traditionally improved by rational approaches and combinatorial methods involving mutagenesis and selection. Recently, other methods have emerged, such as the use of artificial transcription factors and engineering of the native ones. As methods for generating genetic diversity continue to proliferate, the need for quantifying phenotypic diversity and, hence, assessing the potential of various genetic libraries for strain improvement becomes more pronounced. Here, we present a metric based on the quantification of phenotypic diversity, using Lactobacillus plantarum as a model organism. We found that phenotypic diversity can be introduced by mutagenesis of the principal σ factor, that this diversity can be modulated by tuning the sequence diversity, and that this method compares favorably with commonly used protocols for chemical mutagenesis. The results of the diversity metric here developed also correlated well with the probability of finding improved mutants in the different libraries, as determined by recursive screening under stress. In addition, we subjected our libraries to lactic and inorganic acids and found strains with improved growth in both conditions, with a concomitant increase in lactate productivity.

Abstract Monitoring cellular behavior and eventually properly adapting cellular processes is key to handle the enormous complexity of today’s metabolic engineering questions. Hence, transcriptional biosensors bear the potential to augment and accelerate current metabolic engineering strategies, catalyzing vital advances in industrial biotechnology. The development of such transcriptional biosensors typically starts with exploring nature’s richness. Hence, in a first part, the transcriptional biosensor architecture and the various modi operandi are briefly discussed, as well as experimental and computational methods and relevant ontologies to search for natural transcription factors and their corresponding binding sites. In the second part of this review, various engineering approaches are reviewed to tune the main characteristics of these (natural) transcriptional biosensors, i.e., the response curve and ligand specificity, in view of specific industrial biotechnology applications, which is illustrated using success stories of transcriptional biosensor engineering.

Functional characterization of pseudouridine (Ψ) in mammalian mRNA has been hampered by the lack of a quantitative method that maps Ψ in the whole transcriptome. We report bisulfite-induced deletion sequencing (BID-seq), which uses a bisulfite-mediated reaction to convert pseudouridine stoichiometrically into deletion upon reverse transcription without cytosine deamination. BID-seq enables detection of abundant Ψ sites with stoichiometry information in several human cell lines and 12 different mouse tissues using 10–20 ng input RNA. We uncover consensus sequences for Ψ in mammalian mRNA and assign different ‘writer’ proteins to individual Ψ deposition. Our results reveal a transcript stabilization role of Ψ sites installed by TRUB1 in human cancer cells. We also detect the presence of Ψ within stop codons of mammalian mRNA and confirm the role of Ψ in promoting stop codon readthrough in vivo. BID-seq will enable future investigations of the roles of Ψ in diverse biological processes. Pseudouridine sites in mRNA are detected at base resolution and functionally investigated.