Engineering Non-phosphorylative Shortcut Metabolism to Enable Efficient Biosynthesis of TCA-cycle Derivatives

Converting lignocellulosic feedstocks into useful products presents a significant challenge for biosynthesis. For example, it requires more than 13 steps to convert D-xylose into a-ketoglutarate (a-KG) in TCA cycle. In this work, we report the design of a non-phosphorylative “shortcut” metabolism to convert sugars into a-KG in fewer than 6 steps. BDO, with an annual production over 2.5 million tons, was chosen as our target product to prove the application of this “shortcut” metabolism. Primary substrates used in this work are D-xylose, L-arabinose and galacturonic acid which are main components of non-food agricultural byproducts. Since this “shortcut” pathway was not well characterized previously, we performed a series of growth assays to establish the in vivo enzyme activity in E. coli and characterized the kinetic parameters of important enzymes in the “shortcut” metabolic pathway. Different combinations of the downstream enzymes, 2-keto acid decarboxylases and alcohol dehydrogenases, were screened and protein engineering was performed to improve enzyme specificity. In particular, we have constructed a strain of E. coli capable of producing 12 g/L of BDO from D-xylose and another E.  coli strain that can produce 15.6 g/L BDO from L- arabinose. This study demonstrates the application of an efficient and short metabolic pathway towards the production of TCA-cycle derivatives.