Construction Of Recombinant Corynebacterium Glutamicum For Producing Cadaverine By One Step Method And The Optimization Of Fermentation Conditions

Since the Industrial Revolution of the18th century, heavy consumption of petroleum resources has resulted an rapid production of carbon dioxide, which has contributed to escalating global warming. One solution of this problem is to use polymers derived from renewable resources. About1,200,000tons of polyamides are produced in the world per year, and polyamide6(nylon6) and polyamide66(nylon66) make up90%of these polyamides. If we can produce polyamides from renewable resources, this will be an effective measure against global warming on a worldwide scale. Polyamide66is produced by polymerization using hexamethylenediamine, which is a diamine containing6carbons, and adipic acid, which is a dicarboxylic acid containing6carbons, at a molar ratio of1:1. Pentamethylenediamine containing5carbons, also known as cadaverine, is known to be produced from lysine using lysine decarboxylase, with the structure most similar to hexamethylenediamine. Nylon-56, which is polymerized from cadaverine and adipic acid, which is a dicarboxylic acid containing6carbons, is expected to be the polyamide that is formed from these renewable sources.In order to make C. glutamicum to produce ethanol,it is indispensable to express lysine decarboxylase of Hafnia alvei AS1.1009in the the C. glutamicum TK260512.In our previous job, a2.2kb fragment of Hafnia alvei lysine decarboxylase gene encoding lysine decarboxylase (LDC) was proliferated by polymerase chain reaction by using chromosomal DNA of Hafnia alvei as the template. The obtained Idc fragment was inserted into E. coli/C. glutamicum shuttle vector pXMJ19to construct an expression plasmid pXMJ19-1dc. It was then introduced into C. glutamicum TK26o512via electrotransformation, and a recombinant C. glutamicum TK260512/pXMJ19-ldc was obtained. SDS-PAGE electrophoresis profile of whole cell proteins indicated that lysine decarboxylase was expressed and its molecular weight was of83kD.The fermentation conditions for cadaverine of recombinant strain were invstigated in in250mL shake flask scale. The results showed:(1) Culture medium:glucose11%、(NH4)2SO44%、solution of amino acid mixture prepared from soybean hydrolysate5%, CH3COOH0.6%, K2HPO42.5g/L, MgSO4·7H2O0.75g/L, CaCl2·2H2O50mg/L, FeSO4·7H2O50mg/L MnSO4·5H2O13mg/L, nicotinic acid14mg/L, thiamine·HCl7mg/L, D-biotin0.5mg/L, pyridoxal-5’-phosphate0.5mg/L。(pH7.0).(2) Inoculum size:8%(3) Volume of medium:30mL/250mL triangular flask (4) Rotation speed:200r/min Following above conditions, the recombinant stain could produce2.52g/L cadaverine after36h cultivation.83%increased cadaverine yield after optimization of fermentation medium.