Recombinant E. coli idhA protein (a.a. 1-330) [His]

Enterobacter aerogenes was metabolically engineered for acetoin production. To remove the pathway enzymes that catalyzed the formation of by-products, the three genes encoding a lactate dehydrogenase (IdhA) and two 2,3-butanediol dehydrogenases (budC, and dhaD), respectively, were deleted from the genome. The acetoin production was higher under highly aerobic conditions. However, an extracellular glucose oxidative pathway in E. aerogenes was activated under the aerobic conditions, resulting in the accumulation of 2-ketogluconate. To decrease the accumulation of this by-product, the gene encoding a glucose dehydrogenase (gcd) was also deleted. The resulting strain did not produce 2-ketogluconate but produced significant amounts of acetoin, with concentration reaching 71.7 g/L with 2.87 g/L/h productivity in fed-batch fermentation. This result demonstrated the importance of blocking the glucose oxidative pathway under highly aerobic conditions for acetoin production using E. aerogenes.

Haem has widespread applications in healthcare and food supplement industries. Escherichia coli has previously been engineered to produce a small amount of haem intracellularly through the C4 pathway, requiring extraction for applications. Here we report secretory production of free haem by engineered E. coli strains, using the C5 pathway and the optimized downstream pathway for haem biosynthesis. Furthermore, knocking out IdhA, pta and also yfeX-encoding a putative haem-degrading enzyme-results in 7.88 mgl(-1) of total haem with 1.26 mgl(-1) of extracellular haem in flask cultivation. Fed-batch fermentations of the engineered strain overexpressing a haem exporter CcmABC from glucose only and glucose supplemented with L-glutamate secrete 73.4 and 151.4 mgl(-1) of haem, respectively, which are 63.5% of 115.5 mgl(-1) and 63.3% of 239.2 mgl(-1) of total haem produced. The engineered E. coli strain reported here will be useful for microbial production of free haem.