Homologous overexpression of Hydrogenase and Glycerol dehydrogenase in Clostridium pasteurianum to enhance hydrogen production from crude glycerol Shyamali Sarma 1 , David Ortega 2 , Nigel P. Minton 2 , Vikash Kumar Dubey 1# and Vijayanand S. Moholkar 1,* 1 Centre for Energy, Indian Institute of Technology Guwahati, Guwahati – 781 039, Assam # School of Biochemical Engineering, Indian Institute of Technology (BHU), Varanasi-221005 2 Clostridia Research Group, BBSRC/EPSRC Synthetic Biology Research Centre (SBRC), University of Nottingham, Nottingham–NG72RD, United Kingdom * Author for correspondence. E-mail: vmoholkar@iitg.ernet.in, Phone: +91 361 258 2300, Fax: +91 361 258 2291 Abstract This study reports engineering of a hypertransformable variant of C. pasteurianum for bioconversion of glycerol into hydrogen (H2). A functional glycerol-triggered hydrogen pathway was engineered based on two approaches: (1) increasing product yield by overexpression of immediate enzyme catalyzing H2 production, (2) increasing substrate uptake by overexpression of enzymes involved in glycerol utilization. The first strategy aimed at overexpression of hydA gene encoding hydrogenase, and the second one, through combination of overexpression of dhaD1 and dhaK genes encoding glycerol dehydrogenase and dihydroxyacetone kinase. These genetic manipulations resulted in two recombinant strains (hydA ++ /dhaD1K ++ ) capable of producing 97% H2 (v/v), with yields of 1.1 mol H2/mol glycerol in hydA overexpressed strain, and 0.93 mol H2/mol glycerol in dhaD1K overexpressed strain, which was 1.5 fold higher than wild type. Among two strains, dhaD1K ++ consumed more glycerol than hydA ++ which proves that overexpression of glycerol enzymes has enhanced glycerol intake rate. Keywords: Biohydrogen, Crude glycerol, Clostridium pasteurianum, Overexpression