Bioproduction of trans-10,cis-12-Conjugated Linoleic Acid by a Highly Soluble and Conveniently Extracted Linoleic Acid Isomerase and an Extracellularly Expressed Lipase from Recombinant Escherichia coli Strains
2018
Mengnan Huang, Jiangnan University, Wuxi, China | Xinyao Lu, Jiangnan University, Wuxi, China | Hong Zong, Jiangnan University, Wuxi, China | Bin Zhuge, Jiangnan University, Wuxi, China | Wei Shen, Jiangnan University, Wuxi, China
The low solubility and high-cost recovery of Propionibacterium acnes polyunsaturated fatty acid isomerase (PAI) are key problems in the bioproduction of high value-added conjugated linoleic acid (CLA). To improve the solubility of recombinant PAI, six chaperone proteins were coexpressed with PAI. Introduction of GroELS proteins dramatically improved the PAI solubility from 29% to 97%, with increased activity by 57.8%. Combined expression of DnaKJGrpE and GroELS proteins increased the activity by 11.9%. In contrast, coexpression of DnaKJGrpE proteins significantly reduced the activity by 57.4%. Plasmids pTf16 harboring the tig gene and pG-Tf2 containing the tig and groEL-groES genes had no visible impact on PAI expression. The lytic protein E was then introduced into the recombinant Escherichia coli to develop a cell autolysis system. A 35% activity of total intracellular PAI was released from the cytoplasm by suspending the lysed cells in distilled water. The PAI recovery was further improved to 81% by optimizing the release conditions. The lipase from Rhizopus oryzae was also expressed in E. coli, with an extracellular activity of 110.9 U/ml. By using the free PAI and lipase as catalysts, a joint system was established for producing CLA from sunflower oil. Under the optimized conditions, the maximum titer of t-10,c-12-CLA reached 9.4 g/l. This work provides an effective and low-cost strategy to improve the solubility and recovery of the recombinant intracellular PAI for further large-scale production of CLA.
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