CRISPRi screen for enhancing heterologous α-amylase yield in Bacillus subtilis.

Yield improvements in cell factories can potentially be obtained by fine-tuning the regulatory mechanisms for gene candidates. In pursuit of such candidates, we performed RNA-sequencing of two α-amylase producing Bacillus strains and predict hundreds of putative novel non-coding transcribed regions. Surprisingly, we found among hundreds of non-coding and structured RNA candidates that non-coding genomic regions are proportionally undergoing the highest changes in expression during fermentation. Since these classes of RNA are also understudied, we targeted the corresponding genomic regions with CRIPSRi knockdown to test for any potential impact on the yield. From differentially expression analysis, we selected 53 non-coding candidates. Although CRISPRi knockdowns target both the sense and the antisense strand, the CRISPRi experiment cannot link causes for yield changes to the sense or antisense disruption. Nevertheless, we observed on several instances with strong changes in enzyme yield. The knockdown targeting the genomic region for a putative antisense RNA of the 3' UTR of the skfA-skfH operon led to a 21% increase in yield. In contrast, the knockdown targeting the genomic regions of putative antisense RNAs of the cytochrome c oxidase subunit 1 (ctaD), the sigma factor sigH, and the uncharacterized gene yhfT decreased yields by 31 to 43%.

The impact of PrsA over-expression on the Bacillus subtilis transcriptome during fed-batch fermentation of alpha-amylase production.

The production of the alpha-amylase (AMY) enzyme in Bacillus subtilis at a high rate leads to the accumulation of unfolded AMY, which causes secretion stress. The over-expression of the PrsA chaperone aids enzyme folding and reduces stress. To identify affected pathways and potential mechanisms involved in the reduced growth, we analyzed the transcriptomic differences during fed-batch fermentation between a PrsA over-expressing strain and control in a time-series RNA-seq experiment. We observe transcription in 542 unannotated regions, of which 234 had significant changes in expression levels between the samples. Moreover, 1,791 protein-coding sequences, 80 non-coding genes, and 20 riboswitches overlapping UTR regions of coding genes had significant changes in expression. We identified putatively regulated biological processes via gene-set over-representation analysis of the differentially expressed genes; overall, the analysis suggests that the PrsA over-expression affects ATP biosynthesis activity, amino acid metabolism, and cell wall stability. The investigation of the protein interaction network points to a potential impact on cell motility signaling. We discuss the impact of these highlighted mechanisms for reducing secretion stress or detrimental aspects of PrsA over-expression during AMY production.

The Bacillaceae-1 RNA motif comprises two distinct classes.

Non-coding RNAs are key regulatory players in bacteria. Many computationally predicted non-coding RNAs, however, lack functional associations. An example is the Bacillaceae-1 RNA motif, whose Rfam model consists of two hairpin loops. We find the motif conserved in nine of 13 non-pathogenic strains of the genus Bacillus but only in one pathogenic strain. To elucidate functional characteristics, we studied 118 hits of the Rfam model in 11 Bacillus spp. and found two distinct classes based on the ensemble diversity of their RNA secondary structure and the genomic context concerning the ribosomal RNA (rRNA) cluster. Forty hits are associated with the rRNA cluster, of which all 19 hits upstream flanking of 16S rRNA have a reverse complementary structure of low structural diversity. Fifty-two hits have large ensemble diversity, of which 38 are located between two coding genes. For eight hits in Bacillus subtilis, we investigated public expression data under various conditions and observed either the forward or the reverse complementary motif expressed. Five hits are associated with the rRNA cluster. Four of them are located upstream of the 16S rRNA and are not transcriptionally active, but instead, their reverse complements with low structural diversity are expressed together with the rRNA cluster. The three other hits are located between two coding genes in non-conserved genomic loci. Two of them are independently expressed from their surrounding genes and are structurally diverse. In summary, we found that Bacillaceae-1 RNA motifs upstream flanking of ribosomal RNA clusters tend to have one stable structure with the reverse complementary motif expressed in B. subtilis. In contrast, a subgroup of intergenic motifs has the thermodynamic potential for structural switches.

Genome-scale analysis of the high-efficient protein secretion system of Aspergillus oryzae.

BACKGROUND: The koji mold, Aspergillus oryzae is widely used for the production of industrial enzymes due to its particularly high protein secretion capacity and ability to perform post-translational modifications. However, systemic analysis of its secretion system is lacking, generally due to the poorly annotated proteome. RESULTS: Here we defined a functional protein secretory component list of A. oryzae using a previously reported secretory model of S. cerevisiae as scaffold. Additional secretory components were obtained by blast search with the functional components reported in other closely related fungal species such as Aspergillus nidulans and Aspergillus niger. To evaluate the defined component list, we performed transcriptome analysis on three α-amylase over-producing strains with varying levels of secretion capacities. Specifically, secretory components involved in the ER-associated processes (including components involved in the regulation of transport between ER and Golgi) were significantly up-regulated, with many of them never been identified for A. oryzae before. Furthermore, we defined a complete list of the putative A. oryzae secretome and monitored how it was affected by overproducing amylase. CONCLUSION: In combination with the transcriptome data, the most complete secretory component list and the putative secretome, we improved the systemic understanding of the secretory machinery of A. oryzae in response to high levels of protein secretion. The roles of many newly predicted secretory components were experimentally validated and the enriched component list provides a better platform for driving more mechanistic studies of the protein secretory pathway in this industrially important fungus.

Characterization of the Aspergillus niger prtT, a unique regulator of extracellular protease encoding genes.

Expression of several Aspergillus niger genes encoding major secreted, but not vacuolar, protease genes including the major acid protease gene pepA, was shown to be affected in the previously isolated A. niger protease mutant, AB1.13 [Mattern, I.E., van Noort, J.M., van den Berg, P., Archer, D.A., Roberts, I.N., Hondel, C.A.M.J.J., 1992. Isolation and characterization of mutants of Aspergillus niger deficient in extracellular proteases. Molecular & General Genetics 2, 332-336]. Complementation cloning of the putative protease-regulatory gene affected in this mutant was accomplished using a functional selection approach based on the use of the A. nidulans amdS selection marker driven by the A. niger pepA promoter. As expected the PpepA::amdS selection marker is not expressed in the mutant. Introduction of a self-replicating cosmid library into the mutant strain carrying the PpepA::amdS marker allowed selection of AmdS+ transformants functionally complementing the proposed regulatory mutation. Analysis of complementing cosmid clones revealed that the complementing sequences contained a gene encoding a member of the fungal-specific Zn2Cys6-binuclear cluster protein family. Sequence comparison of the encoded protein, PrtT, showed that it has homologues among different Aspergillus species. The A. oryzae homologue was shown to govern expression of the major alkaline protease AlpA and neutral protease Np1 in this species. In contrast to several other pathway specific regulators, such as AmyR and XlnR, no PrtT orthologues could be found in any other non-Aspergillus (or related) species and surprisingly, also not in Aspergillus nidulans. Interestingly, in all Aspergillus species carrying a prtT orthologue the gene is tightly clustered to a completely syntenous region carrying an amylolytic gene cluster including another Zn2Cys6-binuclear cluster protein, AmyR. Northern analysis of the A. niger prtT gene showed (constitutive) expression from two upstream promoters about 700 bp apart. The presence of several short upstream open reading frames downstream of both the distal and the proximal transcription start point of the prtT gene suggests regulation at the post-translational level. Also regulation at the level of differential splicing is suggested by the fact that several Aspergillus EST databases carry a considerable fraction of clones in which in frame intron sequences are retained.

Altering the expression of two chitin synthase genes differentially affects the growth and morphology of Aspergillus oryzae.

In Aspergillus oryzae, one full-length chitin synthase (chsB) and fragments of two other chitin synthases (csmA and chsC) were identified. The deduced amino acid sequence of chsB was similar (87% identity) to chsB from Aspergillus nidulans, which encodes a class III chitin synthase. The sequence obtained for csmA indicated that it had high similarity to class V chitin synthases. chsB and csmA disruption strains and a strain in which chsB transcription was controlled were constructed using the nitrite reductase (niiA) promoter. The strains were examined during hyphal growth by Northern analysis, analysis of the cell-wall composition and growth in the presence of Calcofluor white (CFW). The chsB disrupted strain and the uninduced p(niiA)-chsB strain exhibited hyperbranching, they had a lower level of conidiation than the wild-type and were sensitive to CFW at 50 mg l(-1). When chsB transcription was induced in the strain containing the p(niiA)-chsB construct, the strain displayed wild-type morphology on solid medium and at sub-maximum growth rates but the wild-type morphology was not fully restored during rapid growth in batch cultivation. The csmA disruption strain displayed morphological abnormalities, such as ballooning cells, intrahyphal hyphae and conidial scars. The growth was severely inhibited in the presence of 10 mg CFW l(-1). In none of the constructed strains did the cell-wall composition differ from the wild-type. Northern analysis indicated no change in the transcription of the chitin synthase genes csmA and chsC when chsB expression was altered, and there was no change in the transcription of chsB and chsC when csmA was disrupted.