Microbial Biotechnology最新文献

Exporter engineering is a promising strategy to construct high-yield Streptomyces for natural product pharmaceuticals in industrial biotechnology. However, available exporters are scarce, due to the limited knowledge of bacterial transporters. Here, we built a workflow for exporter mining and devised a tunable plug-and-play exporter (TuPPE) module to improve the production of macrolide biopesticides in Streptomyces. Combining genome analyses and experimental confirmations, we found three ATP-binding cassette transporters that contribute to milbemycin production in Streptomyces bingchenggensis. We then optimized the expression level of target exporters for milbemycin titer optimization by designing a TuPPE module with replaceable promoters and ribosome binding sites. Finally, broader applications of the TuPPE module were implemented in industrial S. bingchenggensis BC04, Streptomyces avermitilis NEAU12 and Streptomyces cyaneogriseus NMWT1, which led to optimal titer improvement of milbemycin A3/A4, avermectin B_1a and nemadectin α by 24.2%, 53.0% and 41.0%, respectively. Our work provides useful exporters and a convenient TuPPE module for titer improvement of macrolide biopesticides in Streptomyces. More importantly, the feasible exporter mining workflow developed here might shed light on widespread applications of exporter engineering in Streptomyces to boost the production of other secondary metabolites.

The development of synthetic biology has brought an unprecedented increase in the number molecular tools applicable into a microbial chassis. The exploration of such tools into different bacteria revealed not only the challenges of context dependency of biological functions but also the complexity and diversity of regulatory layers in bacterial cells. Most of the standardized genetic tools and principles/functions have been mostly based on model microorganisms, namely Escherichia coli. In contrast, the non-model pseudomonads lack a deeper understanding of their regulatory layers and have limited molecular tools. They are resistant pathogens and promising alternative bacterial chassis, making them attractive targets for further studies. Ribonucleases (RNases) are key players in the post-transcriptional control of gene expression by degrading or processing the RNA molecules in the cell. These enzymes act according to the cellular requirements and can also be seen as the recyclers of ribonucleotides, allowing a continuous input of these cellular resources. This makes these post-transcriptional regulators perfect candidates to regulate microbial physiology. This review summarizes the current knowledge and unique properties of ribonucleases in the world of pseudomonads, taking into account genomic context analysis, biological function and strategies to use ribonucleases to improve biotechnological processes.

Secreted proteins and peptides hold large potential both as therapeutics and as enzyme catalysts in biotechnology. The high stability of many secreted proteins helps maintain functional integrity in changing chemical environments and is a contributing factor to their commercial potential. Disulphide bonds constitute an important post-translational modification that stabilizes many of these proteins and thus preserves the active state under chemically stressful conditions. Despite their importance, the discovery and applications within this group of proteins and peptides are limited by the availability of synthetic biology tools and heterologous production systems that allow for efficient formation of disulphide bonds. Here, we refine the design of two DisCoTune (Disulphide bond formation in E. coli with tunable expression) plasmids that enable the formation of disulphides in the highly popular Escherichia coli T7 protein production system. We show that this new system promotes significantly higher yield and activity of an industrial protease and a conotoxin, which belongs to a group of disulphide-rich venom peptides from cone snails with strong potential as research tools and pharmacological agents.

We urgently need new antibiotics to counteract the rising in the emergence of multidrug-resistant microorganisms. To improve the identification of antimicrobial compounds of microbial origin, numerous multidisciplinary approaches are being implemented. However, the development of innovative microbial cultivation strategies is necessary to exploit the full biosynthetic potential of non-culturable microorganisms. Here, I highlight various articles that employ high-throughput microfluidic-based strategies to identify novel antimicrobial metabolites based on bacterial activities. The rapid development of this technology will likely advance the field of antibiotic discovery.

Seventeen species of fungi belonging to thirteen genera were screened for the ability to carry out the transformation of 7-oxo-DHEA (7-oxo-dehydroepiandrosterone). Some strains expressed new patterns of catalytic activity towards the substrate, namely 16β-hydroxylation (Laetiporus sulphureus AM498), Baeyer–Villiger oxidation of ketone in D-ring to lactone (Fusicoccum amygdali AM258) and esterification of the 3β-hydroxy group (Spicaria divaricata AM423). The majority of examined strains were able to reduce the 17-oxo group of the substrate to form 3β,17β-dihydroxy-androst-5-en-7-one. The highest activity was reached with Armillaria mellea AM296 and Ascosphaera apis AM496 for which complete conversion of the starting material was achieved, and the resulting 17β-alcohol was the sole reaction product. Two strains of tested fungi were also capable of stereospecific reduction of the conjugated 7-keto group leading to 7β-hydroxy-DHEA (Inonotus radiatus AM70) or a mixture of 3β,7α,17β-trihydroxy-androst-5-ene and 3β,7β,17β-trihydroxy-androst-5-ene (Piptoporus betulinus AM39). The structures of new metabolites were confirmed by MS and NMR analysis. They were also examined for their cholinesterase inhibitory activity in an enzymatic-based assay in vitro test.

It has been established that gut microbiota influences chicken growth performance and fat metabolism. However, whether gut microbiota affects chicken growth performance by regulating fat metabolism remains unclear. Therefore, seven-week-old chickens with high or low body weight were used in the present study. There were significant differences in body weight, breast and leg muscle indices, and cross-sectional area of muscle cells, suggesting different growth performance. The relative abundance of gut microbiota in the caecal contents at the genus level was compared by 16S rRNA gene sequencing. The results of LEfSe indicated that high body weight chickens contained Microbacterium and Sphingomonas more abundantly (P < 0.05). In contrast, low body weight chickens contained Slackia more abundantly (P < 0.05). The results of H & E, qPCR, IHC, WB and blood analysis suggested significantly different fat metabolism level in serum, liver, abdominal adipose, breast and leg muscles between high and low body weight chickens. Spearman correlation analysis revealed that fat metabolism positively correlated with the relative abundance of Microbacterium and Sphingomonas while negatively correlated with the abundance of Slackia. Furthermore, faecal microbiota transplantation was performed, which verified that transferring faecal microbiota from adult chickens with high body weight into one-day-old chickens improved growth performance and fat metabolism in liver by remodelling the gut microbiota. Overall, these results suggested that gut microbiota could affect chicken growth performance by regulating fat metabolism.

Exorbitant outputs of waste xylose mother liquor (WXML) and corncob residue from commercial-scale production of xylitol create environmental problems. To reduce the wastes, a Saccharomyces cerevisiae strain tolerant to WXML was conferred with abilities to express the genes of xylose reductase, a xylose-specific transporter and enzymes of the pentose phosphate pathway. This strain showed a high capacity to produce xylitol from xylose in WXML with glucose as a co-substrate. Additionally, a simultaneous saccharification and fermentation (SSF) process was designed to use corncob residues and cellulase instead of directly adding glucose as a co-substrate. Xylitol titer and the productivity were, respectively, 91.0 g l^-1 and 1.26 ± 0.01 g l^-1 h^-1 using 20% WXML, 55 g DCW l^-1 delignified corncob residues and 11.8 FPU g_cellulose^-1 cellulase at 35° during fermentation. This work demonstrates the promising strategy of SSF to exploit waste products to xylitol fermentation process.

The lambda phage Red proteins Redα/Redβ/Redγ and Rac prophage RecE/RecT proteins are powerful tools for precise and efficient genetic manipulation but have been limited to only a few prokaryotes. Here, we report the development and application of a new recombineering system for Burkholderia glumae and Burkholderia plantarii based on three Rac bacteriophage RecET-like operons, RecEThe_BDU8, RecETh_TJI49 and RecETh1h2e_YI23, which were obtained from three different Burkholderia species. Recombineering experiments indicated that RecETh_TJI49 and RecETh1h2e_YI23 showed higher recombination efficiency compared to RecEThe_BDU8 in Burkholderia glumae PG1. Furthermore, all of the proteins currently categorized as hypothetical proteins in RecETh1h2e_YI23, RecETh_TJI49 and RecEThe_BDU8 may have a positive effect on recombination in B. glumae PG1 except for the h2 protein in RecETh1h2e_YI23. Additionally, RecET_YI23 combined with exonuclease inhibitors Pluγ or Redγ exhibited equivalent recombination efficiency compared to Redγβα in Escherichia coli, providing potential opportunity of recombineering in other Gram-negative bacteria for its loose host specificity. Using recombinase-assisted in situ insertion of promoters, we successfully activated three cryptic non-ribosomal peptide synthetase biosynthetic gene clusters in Burkholderia strains, resulting in the generation of a series of lipopeptides that were further purified and characterized. Compound 7 exhibited significant potential anti-inflammatory activity by inhibiting lipopolysaccharide-stimulated nitric oxide production in RAW 264.7 macrophages. This recombineering system may greatly enhance functional genome research and the mining of novel natural products in the other species of the genus Burkholderia after optimization of a protocol.