Isopropyl-β-D-thiogalactopyranoside

Cellulosomes are synthesized by anaerobic bacteria and fungi to degrade lignocellulose via synergistic action of multiple enzymes fused to a protein scaffold. Through templating key protein domains (cohesin and dockerin), designer cellulosomes have been engineered from bacterial motifs to alter the activity, stability, and degradation efficiency of enzyme complexes. Recently a parts list for fungal cellulosomes from the anaerobic fungi (Neocallimastigomycota) was determined, which revealed sequence divergent fungal cohesin, dockerin, and scaffoldin domains that could be used to expand the available toolbox to synthesize designer cellulosomes. In this work, multi-domain carbohydrate active enzymes (CAZymes) from 3 cellulosome-producing fungi were analyzed to inform the design of chimeric proteins for synthetic cellulosomes inspired by anaerobic fungi. In particular,Piromyces finniswas used as a structural template for chimeric carbohydrate active enzymes. Recombinant enzymes with retained properties were engineered by combining thermophilic glycosyl hydrolase domains fromThermotoga maritimawith dockerin domains fromPiromyces finnis. By preserving the protein domain order fromP. finnis, chimeric enzymes retained catalytic activity at temperatures over 80 °C and were able to associate with cellulosomes purified from anaerobic fungi. Fungal cellulosomes harbor a wide diversity of glycoside hydrolases, each representing templates for the design of chimeric enzymes. By conserving dockerin domain position within the primary structure of each protein, the activity of both the catalytic domain and dockerin domain was retained in enzyme chimeras. Taken further, the domain positioning inferred from native fungal cellulosome proteins can be used to engineer multi-domain proteins with non-native favorable properties, such as thermostability.

Corynebacterium glutamicumis one of the important industrial microorganisms for production of amino acids and other value-added compounds. Most expression vectors used inC. glutamicumare based on inducible promoter (Ptacor Ptrc) activated by isopropyl-β-D-thiogalactopyranoside (IPTG). However, these vectors seem unsuitable for large-scale industrial production due to the high cost and toxicity of IPTG. Myo-inositol is an ideal inducer because of its non-toxicity and lower price. In this study, a myo-inositol-inducible expression vector pMI-4, derived from the expression vector pXMJ19, was constructed. Besides the original chloramphenicol resistance genecat, multiple cloning sites, andrrnBterminator, the pMI-4 (6,643 bp) contains theiolRqcassette and the myo-inositol-inducible promoter PiolT1. The pMI-4 could stably replicate in theC. glutamicumhost. Meanwhile, the non-myo-inositol degradation host strainC. glutamicumΔiolGΔoxiCΔoxiDΔoxiEfor maintaining the pMI-4 was developed. Overexpression ofhemAMandhemLusing pMI-4 resulted in a significant accumulation of 5-aminolevulinic acid, indicating its potential application in metabolic engineering and industrial fermentation.

Cellular senescence is a unique process of normal physiology, from embryonic development to aging, also known for its association with a broad range of pathological conditions. Therefore a reliable model of cellular senescence remains an indispensable tool for the investigation of senescence-associated changes and human disease. Here we describe a model of HT1080 fibrosarcoma cells with an inducible senescence phenotype. These cells are equipped with the lac repressor and exogenous p21 under the control of a lac repressor regulated promoter. The senescent phenotype is induced in these cells by isopropyl-β-D-thiogalactopyranoside (IPTG)-inducible expression of senescence-associated cell cycle inhibitor p21Waf1/Cip1/Sdi1.