To increase the commercial value of oleaginous fungus Mortierella alpina by incorporation of trans-10,cis-12 conjugated linoleic acid (CLA) into the polyunsaturated fatty acids (PUFAs) of M. alpina via Propionibacterium acnes isomerase (PAI) conversion. The PAI gene and the codon optimization version were heterologously expressed in M. alpina via Agrobacterium tumefaciens-mediated transformation (ATMT). Coding usage modification significantly improved the translation of PAI transcripts and trans-10,cis-12 CLA was produced up to 1.2 mg l(-1), which corresponds to approx. 0.05 % of the total fatty acid (TFA). Since PAI prefers free linoleic acid as a substrate rather than any other forms, 5 mu M long-chain acyl CoA synthetase inhibitor was added and the trans-10,cis-12 CLA content increased approx. 24-fold to 29 mg l(-1), reaching up to 1.2 % (w/w) of the TFA in M. alpina. Heterologous expression of PAI in M. alpina by ATMT methods is a practicable way in biosynthesis of CLA and this system may be a feasible platform for industrial production of CLA.