Development of CRISPR/Cas9-Based Genome Editing Tools for Polyploid Yeast Cyberlindnera jadinii and Its Application in Engineering Heterologous Steroid-Producing Strains.

In this study, a suite of efficient CRISPR/Cas9 tools was developed to overcome the genetic manipulation challenges posed by the polyploid genome of industrial yeast Cyberlindnera jadinii. The developed CRISPR/Cas9 system can achieve a 100% single-gene knockdown efficiency in strain NBRC0988. Moreover, the integration of a single exogenous gene into the target locus using a 50 bp homology arm achieved near-100% efficiency. The efficiency of simultaneous integration of three genes into the chromosome is strongly influenced by the length of the homology arm, with the highest integration efficiency of 62.5% obtained when selecting a homology arm of about 500 bp. By utilizing the CRISPR/Cas system, this study demonstrated the potential of C. jadinii in producing heterologous sterols. Through shake-flask fermentation, the engineered strains produced 92.1 and 81.8 mg/L of campesterol and cholesterol, respectively. Furthermore, the production levels of these two sterols were further enhanced through high-cell-density fed-batch fermentation in a 5 L bioreactor. The highest titer of campesterol reached 807 mg/L [biomass OD600 = 294, productivity of 6.73 mg/(L·h)]. The titer of cholesterol reached 1.52 g/L [biomass OD600 = 380, productivity of 9.06 mg/(L·h)], marking the first gram-scale production of steroidal compounds in C. jadinii.

A Toll pathway effector protects Drosophila specifically from distinct toxins secreted by a fungus or a bacterium.

The Drosophila systemic immune response against many Gram-positive bacteria and fungi is mediated by the Toll pathway. How Toll-regulated effectors actually fulfill this role remains poorly understood as the known Toll-regulated antimicrobial peptide (AMP) genes are active only against filamentous fungi and not against Gram-positive bacteria or yeasts. Besides AMPs, two families of peptides secreted in response to infectious stimuli that activate the Toll pathway have been identified, namely Bomanins and peptides derived from a polyprotein precursor known as Baramicin A (BaraA). Unexpectedly, the deletion of a cluster of 10 Bomanins phenocopies the Toll mutant phenotype of susceptibility to infections. Here, we demonstrate that BaraA is required specifically in the host defense against Enterococcus faecalis and against the entomopathogenic fungus Metarhizium robertsii, albeit the fungal burden is not altered in BaraA mutants. BaraA protects the fly from the action of distinct toxins secreted by these Gram-positive and fungal pathogens, respectively, Enterocin V and Destruxin A. The injection of Destruxin A leads to the rapid paralysis of flies, whether wild type (WT) or mutant. However, a larger fraction of wild-type than BaraA flies recovers from paralysis within 5 to 10 h. BaraAs' function in protecting the host from the deleterious action of Destruxin is required in glial cells, highlighting a resilience role for the Toll pathway in the nervous system against microbial virulence factors. Thus, in complement to the current paradigm, innate immunity can cope effectively with the effects of toxins secreted by pathogens through the secretion of dedicated peptides, independently of xenobiotics detoxification pathways.

Heel pain: A systematic review.

Heel pain is a very common foot disease. Varieties of names such as plantar fasciitis, jogger's heel, tennis heal, policeman's heel are used to describe it. Mechanical factors are the most common etiology of heel pain. Common causes of hell pain includes: Plantar Fasciitis, Heel Spur, Sever's Disease, Heel bump, Achilles Tendinopathy, Heel neuritis, Heel bursitis. The diagnosis is mostly based on clinical examination. Normally, the location of the pain and the absence of associated symptoms indicating a systemic disease strongly suggest the diagnosis. Several therapies exist including rest, physical therapy, stretching, and change in footwear, arch supports, orthotics, night splints, anti-inflammatory agents, and surgery. Almost all patients respond to conservative nonsurgical therapy. Surgery is the last treatment option if all other treatments had failed. Rest, ice, massage, the use of correct exercise and complying with a doctor's advice all play important part in helping to recover from this hell pain condition, but getting good quality, suitable shoes with the appropriate amount of support for the whole foot is the most important.

Fbxo45 inhibits calcium-sensitive proteolysis of N-cadherin and promotes neuronal differentiation.

Fbxo45 is an atypical E3 ubiquitin ligase, which specifically targets proteins for ubiquitin-mediated degradation. Fbxo45 ablation results in defective neuronal differentiation and abnormal formation of neural connections; however, the mechanisms underlying these defects are poorly understood. Using an unbiased mass spectrometry-based proteomic screen, we show here that N-cadherin is a novel interactor of Fbxo45. N-cadherin specifically interacts with Fbxo45 through two consensus motifs overlapping the site of calcium-binding and dimerization of the cadherin molecule. N-cadherin interaction with Fbxo45 is significantly abrogated by calcium treatment. Surprisingly, Fbxo45 depletion by RNAi-mediated silencing results in enhanced proteolysis of N-cadherin. Conversely, ectopic expression of Fbxo45 results in decreased proteolysis of N-cadherin. Fbxo45 depletion results in dramatic reduction in N-cadherin expression, impaired neuronal differentiation, and diminished formation of neuronal processes. Our studies reveal an unanticipated role for an F-box protein that inhibits proteolysis in the regulation of a critical biological process.

Probing conformational changes in T7 RNA polymerase during initiation and termination by using engineered disulfide linkages.

During the transition from an initiation complex to an elongation complex (EC), the single-subunit bacteriophage T7 RNA polymerase (RNAP) undergoes dramatic conformational changes. To explore the significance of these changes, we constructed mutant RNAPs that are able to form disulfide bonds that limit the mobility of elements that are involved in the transition (or its reversal) and examined the effects of the crosslinks on initiation and termination. A crosslink that is specific to the initiation complex conformation blocks transcription at 5-6 nt, presumably by preventing isomerization to an EC. A crosslink that is specific to the EC conformation has relatively little effect on elongation or on termination at a class I terminator (T), which involves the formation of a stable stem-loop structure in the RNA. Crosslinked ECs also pause and resume transcription normally at a class II pause site (concatamer junction) but are deficient in termination at a class II terminator (PTH, which is found in human preparathyroid hormone gene), both of which involve a specific recognition sequence. The crosslinked amino acids in the EC lie close to the upstream end of the RNA-DNA hybrid and may prevent a movement of the polymerase that would assist in displacing or releasing RNA from a relatively unstable DNA-RNA hybrid in the paused PTH complex.

Major conformational changes occur during the transition from an initiation complex to an elongation complex by T7 RNA polymerase.

To examine changes that occur during the transition from an initiation complex (IC) to an elongation complex (EC) in T7 RNA polymerase (RNAP), we used nucleic acid-protein cross-linking methods to probe interactions of the RNAP with RNA and DNA in a halted EC. As the RNA is displaced from the RNA-DNA hybrid approximately 9 bp upstream from the active site (at -9) it interacts with a region within the specificity loop (residues 744-750) and is directed toward a positively charged surface that surrounds residues Lys-302 and Lys-303. Surprisingly, the template and non-template strands of the DNA at the upstream edge of the hybrid (near the site where the RNA is displaced) interact with a region in the N-terminal domain of the RNAP (residues 172-191) that is far away from the specificity loop before isomerization (in the IC). To bring these two regions of the RNAP into proximity, major conformational changes must occur during the transition from an IC to an EC. The observed nucleic acid-protein interactions help to explain the behavior of a number of mutant RNAPs that are affected at various stages in the initiation process and in termination.