Element Of Gene Expression Regulation In Klebsiella Pneumoniae For Production Of 3-Hydroxypropionic ACID

3-hydroxypropionic acid (3-HP) is an important ecomomiacally important platform chemical with broad applications. As a promising host for production of 3-HP, Klebsiella pneumoniae manefists noticeable biochemical attributes, including rapid cell growth, high tolerance to glycerol and powerful ability to metabolize glycerol. Since K. pneumoniae is not a model microorganism, there are few available promoters can be used for genetic engineering which resulting in low glycerol conversion rate and low yield of 3-HP. In this study, we constructed different efficient expression systems which enabled modification of the glycerol metabolic pathways in K. pneumoniae to increase the yield of 3-HP.1?To construct a promoter library, a total of 13 genes associated with growth and metabolism of K. pneumoniae DSM2026 were investigated for their promoter activities with green fluorescent protein gene (GFP) as reporter gene, we found that the promoter of kanamycin-resistance gene (Pkan) exhibited highest transcriptional intensity which are 1.7 fold higher than that of Pk, the native promoter of glycerol dehydrogenase which was previously used for driving gene expression in K. pneumoniae. This newly developed expression system allows efficient expression of desired gene without addition of any inducer. Moreover, this expression system has no influence on bacterial growth.2?The aldehyde dehydrogenase gene aldH was subcloned into the vector under the promoter of kanamycin-resistance gene. Transformation of the vector into K. pneumoniae resulted in a recombinant strain named K. p (pETPkan-aldH). Under micro-aerobic and shake-flask conditions, the recombinant strain produced 0.47 g/L 3-HP, which was 89.5% increase compared with wild-type strain. The glycerol conversion ratio increased by 100%. Micro-aerobic fed-batch fermentation in a 5 L bioreactor showed that the recombinant strain produced 15.28 g/L 3-HP at 27 h.3?The aldehyde dehydrogenase gene puuC from K. pneumoniae was subcloned into vector under the promoter clusters consisted of multiple core-tac-promoter in tandem. Transformation of these vectors into K. pneumoniae respectively resulted in recombinant strains which named K. pneumoniae (pETPncore-tac-puuC) n=1?5. Under micro-aerobic and shake-flask conditions, the recombinant strain K. pneumoniae (pETP3core-tac-puuC) and K. pneumoniae (pETP5core-tac-puuC) produced 2.88 g/L and 3.10 g/L of 3-HP respectively. When cultivated in a 5 L bioreactor, the recombinant strain K. pneumoniae (pETP3core-tac-puuC) produced 99.8 g/L of 3-HP in 96 h with 87.7% glycerol conversion rate. Meanwhile, byproducts lactic acid was significantly attenuated due to the strong metabolic flux toward 3-HP. The recombinant strain K. pneumoniae (pETP5core-tac-puuC) generated up to 81.4 g/L of 3-HP in 60 h with 69.3% glycerol conversion rate and the productivity was 1.04 g·L-1·h-1 and 1.36 g·L-1·h-1,respectively. More importantly, glycerol was almost completely consumed by the end of fermentation. This largly simplifies the downstream separation, which is of great importance for industrial production of 3-HP.