Engineering resistance to phage GVE3 in Geobacillus thermoglucosidasius

Abstract

Geobacillus thermoglucosidasius is a promising platform organism for the production of biofuels and other metabolites of interest. G. thermoglucosidasius fermentations could be subject to bacteriophage-related failure and financial loss. We develop two strains resistant to a recently described G. thermoglucosidasius-infecting phage GVE3. The phage-encoded immunity gene, imm, was overexpressed in the host leading to phage resistance. A phage-resistant mutant was isolated following expression of a putative anti-repressor-like protein and phage challenge. A point mutation was identified in the polysaccharide pyruvyl transferase, csaB. A double crossover knockout mutation of csaB confirmed its role in the phage resistance phenotype. These resistance mechanisms appear to prevent phage DNA injection and/or lysogenic conversion rather than just reducing efficiency of plating, as no phage DNA could be detected in resistant bacteria challenged with GVE3 and no plaques observed even at high phage titers. Not only do the strains developed here shed light on the biological relationship between the GVE3 phage and its host, they could be employed by those looking to make use of this organism for metabolite production, with reduced occurrence of GVE3-related failure.

The in vivo detection and measurement of the unfolded protein response in recombinant cellulase producing Saccharomyces cerevisiae strains

“…This tool will help to identify the best cellulase gene candidates for CBP strain engineering that yield the required activity while not inducing high levels of secretion-based stress. Furthermore, we can also use this biosensor to investigate whether or not enhanced secretion phenotypes observed in previously engineered strains were partly due to decreased levels of UPR induction [9,10,[50][51][52]. These CEN-based plasmids are amenable to high-throughput transformation approaches, in addition to providing more stable biosensors in the variety of natural and industrial strain isolates often used to develop CBP yeast strains [50,53].…”

“…The S. cerevisiae yENO1 reference strain was used in this study as it was a strain in which the UPR was uninduced and was thus used to compare UPR induction levels of recombinant cellulase secreting S. cerevisiae Y294 strains. The S. cerevisiae Y294 CBH and β-glucosidase (BGL) producing strains used here were previously described [9,10,12]. The reporter genes for the UPR biosensor, T.r.xyn2 and eGFP, were selected due to the minimal effect that they had on the yeast cells and their ability to be easily be assayed.…”

“…The yeast Saccharomyces cerevisiae has many desirable traits for CBP such as high ethanol productivity, high ethanol yield, and comparatively high resistance to inhibitors found in the lignocellulosic hydrolysate [6,7]. S. cerevisiae is also an extensively studied host for heterologous protein production and has thus been the subject of much research to engineer and enhance the expression of necessary cellulases to accomplish the ultimate goal of being able to break down lignocellulose for second-generation production of bioethanol [7][8][9][10][11]. Despite significant success achieved in cellulase expression in yeast, the secretion titers of cellobiohydrolases (CBHs) have been relatively low, and one of the reasons for this phenomenon is the induction of the unfolded protein response (UPR) [4,12,13].…”

Bacteriophages of Thermophilic ‘Bacillus Group’ Bacteria—A Review

“…Tetranucleotide usage deviation analysis confirms this finding, indicating that the GVE3 genome sequence correlates mostly with B. anthracis and B. cereus sequences, rather than Geobacillus sp. sequences [ 62 , 63 ], which again points to widespread genetic recombination among meso- and thermophilic ‘ Bacillus group’ bacteriophages. The analysis also suggests that there exist a different ‘natural’ host, unidentified Geobacillus sp., with tetranucleotide usage deviation patterns more similar to the two Bacillus species that might be the “natural” hosts for GVE3.…”