heterologous expression
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Santhana Nakapong ◽  
Suthipapun Tumhom ◽  
Jarunee Kaulpiboon ◽  
Piamsook Pongsawasdi

2021 ◽  
Vol 9 (3) ◽  
Nils Böhringer ◽  
Robert Green ◽  
Yang Liu ◽  
Ute Mettal ◽  
Michael Marner ◽  

Therapeutic options to combat Gram-negative bacterial pathogens are dwindling with increasing antibiotic resistance. This study presents a proof of concept for the heterologous-expression approach to expand on the novel antibiotic class of darobactins and to generate analogs with different activities and pharmacokinetic properties.

Teun Kuil ◽  
Shuen Hon ◽  
Johannes Yayo ◽  
Charles Foster ◽  
Giulia Ravagnan ◽  

The atypical glycolysis of Clostridium thermocellum is characterized by the use of pyrophosphate (PP i ) as phosphoryl donor for phosphofructokinase (Pfk) and pyruvate phosphate dikinase (Ppdk) reactions. Previously, biosynthetic PP i was calculated to be stoichiometrically insufficient to drive glycolysis. This study investigates the role of a H + -pumping membrane-bound pyrophosphatase, glycogen cycling, a predicted Ppdk–malate shunt cycle and acetate cycling in generating PP i . Knockout studies and enzyme assays confirmed that clo1313_0823 encodes a membrane-bound pyrophosphatase. Additionally, clo1313_0717-0718 was confirmed to encode ADP-glucose synthase by knockouts, glycogen measurements in C. thermocellum and heterologous expression in E. coli . Unexpectedly, individually-targeted gene deletions of the four putative PP i sources did not have a significant phenotypic effect. Although combinatorial deletion of all four putative PP i sources reduced the growth rate by 22% (0.30±0.01 h −1 ) and the biomass yield by 38% (0.18±0.00 g biomass g substrate −1 ), this change was much smaller than what would be expected for stoichiometrically essential PP i -supplying mechanisms. Growth-arrested cells of the quadruple knockout readily fermented cellobiose indicating that the unknown PP i -supplying mechanisms are independent of biosynthesis. An alternative hypothesis that ATP-dependent Pfk activity circumvents a need for PP i altogether, was falsified by enzyme assays, heterologous expression of candidate genes and whole-genome sequencing. As a secondary outcome, enzymatic assays confirmed functional annotation of clo1313_1832 as ATP- and GTP-dependent fructokinase. These results indicate that the four investigated PP i sources individually and combined play no significant PP i -supplying role and the true source(s) of PP i , or alternative phosphorylating mechanisms, that drive glycolysis in C. thermocellum remain(s) elusive. IMPORTANCE Increased understanding of the central metabolism of C. thermocellum is important from a fundamental as well as from a sustainability and industrial perspective. In addition to showing that H + -pumping membrane-bound PPase, glycogen cycling, a Ppdk–malate shunt cycle, and acetate cycling are not significant sources of PP i supply, this study adds functional annotation of four genes and availability of an updated PP i stoichiometry from biosynthesis to the scientific domain. Together, this aids future metabolic engineering attempts aimed to improve C. thermocellum as a cell factory for sustainable and efficient production of ethanol from lignocellulosic material through consolidated bioprocessing with minimal pretreatment. Getting closer to elucidating the elusive source of PP i , or alternative phosphorylating mechanisms, for the atypical glycolysis is itself of fundamental importance. Additionally, the findings of this study directly contribute to investigations into trade-offs between thermodynamic driving force versus energy yield of PP i - and ATP-dependent glycolysis.

2021 ◽  
Vol 7 (12) ◽  
Adriana Rego ◽  
Antonio Fernandez-Guerra ◽  
Pedro Duarte ◽  
Philipp Assmy ◽  
Pedro N. Leão ◽  

Polyketide synthases (PKSs) and non-ribosomal peptide synthetases (NRPSs) are mega enzymes responsible for the biosynthesis of a large fraction of natural products (NPs). Molecular markers for biosynthetic genes, such as the ketosynthase (KS) domain of PKSs, have been used to assess the diversity and distribution of biosynthetic genes in complex microbial communities. More recently, metagenomic studies have complemented and enhanced this approach by allowing the recovery of complete biosynthetic gene clusters (BGCs) from environmental DNA. In this study, the distribution and diversity of biosynthetic genes and clusters from Arctic Ocean samples (NICE-2015 expedition), was assessed using PCR-based strategies coupled with high-throughput sequencing and metagenomic analysis. In total, 149 KS domain OTU sequences were recovered, 36 % of which could not be assigned to any known BGC. In addition, 74 bacterial metagenome-assembled genomes were recovered, from which 179 BGCs were extracted. A network analysis identified potential new NP families, including non-ribosomal peptides and polyketides. Complete or near-complete BGCs were recovered, which will enable future heterologous expression efforts to uncover the respective NPs. Our study represents the first report of biosynthetic diversity assessed for Arctic Ocean metagenomes and highlights the potential of Arctic Ocean planktonic microbiomes for the discovery of novel secondary metabolites. The strategy employed in this study will enable future bioprospection, by identifying promising samples for bacterial isolation efforts, while providing also full-length BGCs for heterologous expression.

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