Omics analysis coupled with gene editing revealed potential transporters and regulators related to levoglucosan metabolism efficiency of the engineered Escherichia coli

Background Bioconversion of levoglucosan, a promising sugar derived from the pyrolysis of lignocellulose, into biofuels and chemicals can reduce our dependence on fossil-based raw materials. However, this bioconversion process in microbial strains is challenging due to the lack of catalytic enzyme relevant to levoglucosan metabolism, narrow production ranges of the native strains, poor cellular transport rate of levoglucosan, and inhibition of levoglucosan metabolism by other sugars co-existing in the lignocellulose pyrolysate. The heterologous expression of eukaryotic levoglucosan kinase gene in suitable microbial hosts like Escherichia coli could overcome the first two challenges to some extent; however, no research has been dedicated to resolving the last two issues till now. Results Aiming to resolve the two unsolved problems, we revealed that seven ABC transporters (XylF, MalE, UgpB, UgpC, YtfQ, YphF, and MglA), three MFS transporters (KgtP, GntT, and ActP), and seven regulatory proteins (GalS, MhpR, YkgD, Rsd, Ybl162, MalM, and IraP) in the previously engineered levoglucosan-utilizing and ethanol-producing E. coli LGE2 were induced upon exposure to levoglucosan using comparative proteomics technique, indicating these transporters and regulators were involved in the transport and metabolic regulation of levoglucosan. The proteomics results were further verified by transcriptional analysis of 16 randomly selected genes. Subsequent gene knockout and complementation tests revealed that ABC transporter XylF was likely to be a levoglucosan transporter. Molecular docking showed that levoglucosan can bind to the active pocket of XylF by seven H-bonds with relatively strong strength. Conclusion This study focusing on the omics discrepancies between the utilization of levoglucosan and non-levoglucosan sugar, could provide better understanding of levoglucosan transport and metabolism mechanisms by identifying the transporters and regulators related to the uptake and regulation of levoglucosan metabolism. The protein database generated from this study could be used for further screening and characterization of the transporter(s) and regulator(s) for downstream enzymatic/genetic engineering work, thereby facilitating more efficient microbial utilization of levoglucosan for biofuels and chemicals production in future.

Evolution of a New Testis-Specific Functional Promoter Within the Highly Conserved Map2k7 Gene of the Mouse

Map2k7 (synonym Mkk7) is a conserved regulatory kinase gene and a central component of the JNK signaling cascade with key functions during cellular differentiation. It shows complex transcription patterns, and different transcript isoforms are known in the mouse (Mus musculus). We have previously identified a newly evolved testis-specific transcript for the Map2k7 gene in the subspecies M. m. domesticus. Here, we identify the new promoter that drives this transcript and find that it codes for an open reading frame (ORF) of 50 amino acids. The new promoter was gained in the stem lineage of closely related mouse species but was secondarily lost in the subspecies M. m. musculus and M. m. castaneus. A single mutation can be correlated with its transcriptional activity in M. m. domesticus, and cell culture assays demonstrate the capability of this mutation to drive expression. A mouse knockout line in which the promoter region of the new transcript is deleted reveals a functional contribution of the newly evolved promoter to sperm motility and the spermatid transcriptome. Our data show that a new functional transcript (and possibly protein) can evolve within an otherwise highly conserved gene, supporting the notion of regulatory changes contributing to the emergence of evolutionary novelties.

The Combination of Bacillus Thuringiensis and Its Engineered Strain Expressing dsRNA Increases the Toxicity against Plutella Xylostella

RNA interference (RNAi) has been developed and used as an emerging strategy for pest management. Here, an entomopathogen Bacillus thuringiensis (Bt) was used to express the dsRNA for the control of Plutella xylostella. A vector containing a 325-bp fragment of the conserved region of P. xylostella arginine kinase gene (PxAK) flanking in two ends with the promoter Pro3α was developed and transferred into Bt 8010 and BMB171, and consequently engineered Bt strains 8010AKi and BMB171AKi expressing dsRNA of PxAK were developed. The two engineered Bt strains were separately mixed with Bt 8010 in a series of ratios, and then fed to the P. xylostella larvae. We found that 8010:8010AKi of 9:1 and 8010:BMB171AKi of 7:3 caused a higher mortality than Bt 8010. PxAK expression levels in the individuals treated with the mixtures, 8010AKi and BMB171Aki, were lower than that in the control. The intrinsic rate of increase (r) and net reproductive rate (R0) of the population treated with 8010:8010AKi of 9:1 were lower than those of the population treated with Bt 8010 or 8010AKi. We developed a Bt-mediated insect RNAi for the control of P. xylostella and demonstrated a practical approach to integrating the entomopathogen with RNAi technique for the pest management.

Pseudomonas aeruginosa utilises host‐derived polyamines to facilitate antimicrobial tolerance

Pseudomonas aeruginosa undergoes diversification during infection of the cystic fibrosis (CF) lung. Understanding these changes requires model systems that capture the complexity of the CF lung environment. We previously identified loss-of-function mutations in the two-component regulatory system sensor kinase gene pmrB, in P. aeruginosa from CF and from experimental infection of mice. Here, we demonstrate that whilst such mutations lower in vitro MICs for multiple antimicrobial classes, this is not reflected in increased antibiotic susceptibility in vivo. Loss of PmrB impairs aminoarabinose modification of lipopolysaccharide, increasing the negative charge of the outer membrane and promoting uptake of cationic antimicrobials. However, in vivo, this can be offset by increased membrane binding of other positively charged molecules present in lungs. The polyamine spermidine readily coats the surface of PmrB-deficient P. aeruginosa, reducing susceptibility to antibiotics that rely on charge differences to bind the outer membrane and increasing biofilm formation. Spermidine is elevated in lungs during P. aeruginosa infection in mice and during episodes of antimicrobial treatment in people with CF. These findings highlight the need to study antimicrobial resistance under clinically relevant environmental conditions. Microbial mutations carrying fitness costs in vitro may be advantageous during infection, where host resources can be utilised.

Genomic profiling and expression analysis of the diacylglycerol kinase gene family in heterologous hexaploid wheat

The inositol phospholipid signaling system mediates plant growth, development, and responses to adverse conditions. Diacylglycerol kinase (DGK) is one of the key enzymes in the phosphoinositide-cycle (PI-cycle), which catalyzes the phosphorylation of diacylglycerol (DAG) to form phosphatidic acid (PA). To date, comprehensive genomic and functional analyses of DGKs have not been reported in wheat. In this study, 24 DGK gene family members from the wheat genome (TaDGKs) were identified and analyzed. Each putative protein was found to consist of a DGK catalytic domain and an accessory domain. The analyses of phylogenetic and gene structure analyses revealed that each TaDGK gene could be grouped into clusters I, II, or III. In each phylogenetic subgroup, the TaDGKs demonstrated high conservation of functional domains, for example, of gene structure and amino acid sequences. Four coding sequences were then cloned from Chinese Spring wheat. Expression analysis of these four genes revealed that each had a unique spatial and developmental expression pattern, indicating their functional diversification across wheat growth and development processes. Additionally, TaDGKs were also prominently up-regulated under salt and drought stresses, suggesting their possible roles in dealing with adverse environmental conditions. Further cis-regulatory elements analysis elucidated transcriptional regulation and potential biological functions. These results provide valuable information for understanding the putative functions of DGKs in wheat and support deeper functional analysis of this pivotal gene family. The 24 TaDGKs identified and analyzed in this study provide a strong foundation for further exploration of the biological function and regulatory mechanisms of TaDGKs in response to environmental stimuli.

Oligomerization of Mutant p53 R273H is not Required for Gain-of-Function Chromatin Associated Activities

The TP53 gene is often mutated in cancer, with missense mutations found in the central DNA binding domain, and less often in the C-terminal oligomerization domain (OD). These types of mutations are found in patients with the rare inherited cancer predisposition disorder called Li-Fraumeni syndrome. We previously found that mutant p53 (mtp53) R273H associates with replicating DNA and promotes the chromatin association of replication-associated proteins mini-chromosome maintenance 2 (MCM2), and poly ADP-ribose polymerase 1(PARP1). Herein, we created dual mutants in order to test if the oligomerization state of mtp53 R273H played a role in chromatin binding oncogenic gain-of-function (GOF) activities. We used site-directed mutagenesis to introduce point mutations in the OD in wild-type p53 (wtp53), and mtp53 R273H expressing plasmids. The glutaraldehyde crosslinking assay revealed that both wtp53 and mtp53 R273H formed predominantly tetramers, while the single OD mutant A347D, and the dual mtp53 R273H-A347D, formed predominantly dimers. The R337C, L344P, mtp53 R273H-R337C, and mtp53 R273H-L344P proteins formed predominantly monomers. Wtp53 was able to activate the cyclin-dependent kinase gene p21/waf and the p53 feedback regulator MDM2. As expected, the transactivation activity was lost for all the single mutants, as well as the mtp53 R273H-dual mutants. Importantly, mtp53 R273H and the dual oligomerization mutants, R273H-A347D, R273H-R337C, and R273H-L344P were able to interact with chromatin. Additionally, the dual oligomerization mutants, R273H-A347D, R273H-R337C, and R273H-L344P, maintained strong interactions with MCM2 and PARP1. Our findings suggest that while mtp53 R273H can form tetramers, tetramer formation is not required for the GOF associated chromatin interactions.

A multi-enhancer RET regulatory code is disrupted in Hirschsprung disease

The major genetic risk factors for Hirschsprung disease (HSCR) are three common polymorphisms within cis-regulatory elements (CREs) of the receptor tyrosine kinase gene RET, which reduce its expression during enteric nervous system (ENS) development. These risk variants attenuate binding of the transcription factors RARB, GATA2, and SOX10 to their cognate CREs, reduce RET gene expression, and dysregulate other ENS and HSCR genes in the RET–EDNRB gene regulatory network (GRN). Here, we use siRNA, ChIP, and CRISPR-Cas9 deletion analyses in the SK-N-SH cell line to ask how many additional HSCR-associated risk variants reside in RET CREs that affect its gene expression. We identify 22 HSCR-associated variants in candidate RET CREs, of which seven have differential allele-specific in vitro enhancer activity, and four of these seven affect RET gene expression; of these, two enhancers are bound by the transcription factor PAX3. We also show that deleting multiple variant-containing enhancers leads to synergistic effects on RET gene expression. These, coupled with our prior results, show that common sequence variants in at least 10 RET enhancers affect HSCR risk, seven with experimental evidence of affecting RET gene expression, extending the known RET–EDNRB GRN to reveal an extensive regulatory code modulating disease risk at a single gene.