Covid-19: six million doses of hydroxychloroquine donated to US despite lack of evidence
Elisabeth Mahase
Abstract
<h3>Abstract</h3> Type I polyketide synthases (PKSs) are large multi-domain proteins converting simple acyl-CoA thioesters such as acetyl-CoA and malonyl-CoA to a large diversity of biotechnologically interesting molecules. Such multi-step reaction cascades are of particular interest for applications in engineered microbial cell factories, as the introduction of a single protein with many enzymatic activities does not require balancing of several individual enzymatic activities. However, functional introduction of type I PKSs into heterologous hosts is very challenging as the large polypeptide chains often do not fold properly. In addition, PKS usually require post-translational activation by dedicated 4’-phosphopantetheinyl transferases (PPTases). Here, we introduce an engineered <i>Corynebacterium glutamicum</i> strain as a novel microbial cell factory for type I PKS-derived products. Suitability of <i>C. glutamicum</i> for polyketide synthesis could be demonstrated by the functional introduction of the 6-methylsalicylic acid synthase ChlB1 from <i>Streptomyces antibioticus</i>. Challenges related to protein folding could be overcome by translation fusion of ChlB1<sub><i>Sa</i></sub> to the C-terminus of the maltose-binding protein MalE from <i>Escherichia coli</i>. Surprisingly, ChlB1<sub><i>Sa</i></sub> was also active in absence of a heterologous PPTase, which finally led to the discovery that the endogenous PPTase PptA<sub><i>Cg</i></sub> of <i>C. glutamicum</i> can also activate ChlB1<sub><i>Sa</i></sub>. The best strain, engineered to provide increased levels of acetyl-CoA and malonyl-CoA, accumulated up to 41 mg/L (0.27 mM) 6-methylsalicylic acid within 48 h of cultivation. Further experiments showed that PptA<sub><i>Cg</i></sub> of <i>C. glutamicum</i> can also activate nonribosomal peptide synthetases (NRPSs), rendering <i>C. glutamicum</i> a promising microbial cell factory for the production of several fine chemicals and medicinal drugs.