Fine-tuning of gene expression is crucial for protein expression and pathway construction, but it still faces formidable challenges due to the hierarchical gene regulation at multiple levels in a context-dependent manner. In this study, we defined the optimal targeting windows for CRISPRa and CRISPRi of the dCas9-α/ω system, and demonstrated that this system could act as a single master regulator to simultaneously activate and repress the expression of different genes by designing position-specific gRNAs. The application scope of dCas9-ω was further expanded by a newly developed CRISPR-assisted O ligonucleotide A nnealing based P romoter S huffling (OAPS) strategy, which could generate a high proportion of functional promoter mutants and facilitate the construction of effective promoter libraries in microorganisms with low transformation efficiency. Combing OAPS and dCas9-ω, the influences of promoter-based transcription, molecular chaperone-assisted protein folding and protease-mediated degradation on the expression of amylase BLA in Bacillus subtilis were systematically evaluated, and a 260-fold enhancement of BLA production was obtained. The success of the OAPS strategy and dCas9-ω for BLA production in this study thus demonstrated that it could serve as a powerful tool kit to regulate the expression of multiple genes multi-directionally and multi-dimensionally in bacteria.

In the present study, ocular chlamydial infections in pigs that originate from two different farming systems were investigated. In particular, the aim was to test pigs with and without clinical ocular symptoms for the presence of Chlamydiaceae and for linked infections with Acanthamoebae spp. possibly acting as vectors for Chlamydia or Chlamydia-like organisms. In a total of 181 pigs, 102 from Germany (GER), representing the intensively kept animals and 79 from Switzerland (CH), which were kept extensively, were screened for the presence of different pathogens by PCR, including a new Chlamydiaceae-specific intergenic spacer rRNA gene PCR. Additionally, results of clinical examination and cytology were compared between the symptomatic and asymptomatic pigs of the two groups. Ocular symptomatic pigs showed a high prevalence of Chlamydia suis in both groups: CH 79%, GER 90%. Only 23% asymptomatic pigs from CH, but 88% asymptomatic pigs from GER were positive for C. suis by PCR. DNA of Chlamydia-like organisms were detected in 19% CH, but only in 2% GER pigs, whereas only 4% CH and 1% GER pigs were also positive for Acanthamoebae spp. A co-infection of Acanthamoebae spp. and C. suis was present in only 3% of the CH but 28% of the GER pigs. In general, the intensively kept pigs in our study seemed to be pre-disposed to ocular chlamydial infection and associated conjunctivitis. Infections with Chlamydia-like organisms alone and in combination with Acanthamoebae played no role for clinical findings within the tested pig groups, whereas a co-infection of Acanthamoebae and C. suis was able to cause serious ocular manifestations in half of the cases of intensively kept pigs being positive for these microorganisms.

As a common expression host, Escherichia coli has received more and more attention due to the recently developed secretory expression system, which offers advantages like reduced downstream bioprocesses and improved product quality. These advantages, coupled with high-density fermentation technology, make it a preferred system for large-scale production of many proteins utilized in industry and agriculture at a reduced process cost. To improve the secretion efficiency of target proteins, various strategies, including signal peptide optimization, periplasmic leakage, and chaperones co-expression have been developed. In addition, the optimization of the fermentation conditions such as temperature, inducer, and medium were also taken into account for the extracellular production in the high-density fermentation to reduce the cost of production. Here, these strategies ranging from genetic engineering to fermentation optimization were summarized for the future guidance of extracellular production of recombinant proteins using E. coli.

BackgroundFor enzymes with buried active sites, transporting substrates/products ligands between active sites and bulk solvent via access tunnels is a key step in the catalytic cycle of these enzymes. Thus, tunnel engineering is becoming a powerful strategy to refine the catalytic properties of these enzymes. The tunnel-like structures have been described in enzymes catalyzing bulky substrates like glycosyl hydrolases, while it is still uncertain whether these structures involved in ligands exchange. Till so far, no studies have been reported on the application of tunnel engineering strategy for optimizing properties of enzymes catalyzing biopolymers.ResultsIn this study, xylanase S7-xyl (PDB: 2UWF) with a deep active cleft was chosen as a study model to evaluate the functionalities of tunnel-like structures on the properties of biopolymer-degrading enzymes. Three tunnel-like structures in S7-xyl were identified and simultaneously reshaped through multi-sites saturated mutagenesis; the most advantageous mutant 254RL1 (V207N/Q238S/W241R) exhibited 340% increase in specific activity compared to S7-xyl. Deconvolution analysis revealed that all three mutations contributed synergistically to the improved activity of 254RL1. Enzymatic characterization showed that larger end products were released in 254RL1, while substrate binding and structural stability were not changed. Dissection of the structural alterations revealed that both the tun_1 and tun_2 in 254RL1 have larger bottleneck radius and shorter length than those of S7-xyl, suggesting that these tunnel-like structures may function as products transportation pathways. Attributed to the improved catalytic efficiency, 254RL1 represents a superior accessory enzyme to enhance the hydrolysis efficiency of cellulase towards different pretreated lignocellulose materials. In addition, tunnel engineering strategy was also successfully applied to improve the catalytic activities of three other xylanases including xylanase NG27-xyl from Bacillus sp. strain NG-27, TSAA1-xyl from Geobacillus sp. TSAA1 and N165-xyl from Bacillus sp. N16-5, with 80%, 20% and 170% increase in specific activity, respectively.ConclusionsThis study represents a pilot study of engineering and functional verification of tunnel-like structures in enzymes catalyzing biopolymer. The specific activities of four xylanases with buried active sites were successfully improved by tunnel engineering. It is highly likely that tunnel reshaping can be used to engineer better biomass-degrading abilities in other lignocellulolytic enzymes with buried active sites.

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