During the past decades biodiesel (monoalkyl esters of long-chain fatty acids) is receiving increasing attention as an alternative diesel fuel because of its favorable properties, environmental benefits and the fact that is derived from the renewable biological resources [1, 2]. At present, biodiesel is mainly produced from edible oils (more than 95%), such as soybean oil, rapeseed oil and palm oil, which leads to global imbalance to the food supply and demand market. One alternative way is to produce biodiesel from low cost non-edible oils. Most of the non-edible plants can be grown in wasteland and infertile land, which allows the use of wasteland to produce oil crops for biodiesel production without the need to compete with food crops for the limited arable land [3, 4]. Thus, focus should be shifted to the sustainable non-edible resources which will be crucial determinants in the popularization of biodiesel.
Tung oil, called “China wood oil” is one kind of promising non-edible biodiesel oil in China. Tung trees are spread widely in China. The oil content of the seeds and whole nuts is approximately 21 and 41 wt.%, respectively. The productivity of tung oil is from 300 to 450 kg/ha, obtained by pressing the seeds of the tung tree. Unlike vegetable oils that contain high amounts of saturated fatty acids, tung oil is composed of more than 60% unsaturated fatty acid, mainly 9Z,11E,13E-α-elaeostearic acid [5, 6]. Generally, saturated fatty acid methyl esters have good oxidation stability and poor low temperature properties. On the contrary, unsaturated fatty acid methyl esters have good low temperature properties and poor oxidation stability . The shortcomings of tung oil methyl esters could be solved by blending with palm, coconut and canola oil biodiesels [5, 7].
Biodiesel could be produced by chemical or enzymatic methods according to the catalysts employed in the process. Contrary to chemical catalysts, enzymatic method does not form soaps and can esterify both FFA and TAG in one step without the need of a subsequent washing step [2, 8]. This is especially the case when using feeds high in FFA such as tung oil. To our knowledge, tung oil has not been used to produce biodiesel by enzymatic method, except for several reports by chemical method [6, 7, 9, 10].
Nowadays biodiesel production by lipase-catalyzed transesterification becomes an interesting prospect in an industrial scale. The enzymatic production of biodiesel has been investigated extensively by using Rhizopus oryzae lipase (ROL) [11, 12]. The free R. oryzae lipase F-APl5 (Amano) catalyzed the methanolysis of soybean oil which reached 80 wt.% yield of fatty acid methyl esters (FAME) by stepwise additions of methanol to the reaction mixture in the presence of 4~30 wt.% water . The crude recombinant R. oryzae lipase LY6 by Pichia pastoris immobilized on anion exchange resin Amberlite IRA-93 was used to biosynthesis biodiesel from soybean oil and the highest biodiesel yield was achieved at 90.5% .
However, the high cost of the catalyst ROL remains a barrier for its industrial applications. In order to bring the cost down, one of the options is to enhance the expression level of R. oryzae lipases. The production of active R. oryzae lipases has been performed in Escherichia coli, in Saccharomyces cerevisiae[16, 17] and in P. pastoris[18–20]. Lorenzo et al. successfully expressed the R. oryzae prolipase (proROL) in a soluble form in E. coli with an activity of 166 U/mL (protein concentration 1.5 mg/mL). Takahashi et al. reported that the activity of proROL by S. cerevisiae reached 2.88 U/mL (protein concentration 28.0 mg/L, OD
600 about 90) after 120 h of cultivation in YPD medium. The activity of R. arrhizus prolipase expressed in P. pastoris was obtained at 140 U/mL (4 375 U/g dry cell weight and 91 mg enzyme/L broth) after 92 h of cultivation in complex medium . The activity of the mature lipase from R. oryzae (mROL) expressed in P. pastoris reached 500 U/mL (60 g wet cell weight/L and 60 mg enzyme/L) . And the activity of this lipase was further improved to 1334 U/mL (about 48 g dry cell weight/L) by a methanol feeding strategy , which was the highest expression level ever reported. However, through different heterologous expression strategies and fermentation techniques, the expression level of R. oryzae lipases is still not optimistic for industry applications.
The prosequenes in some proteolytic enzyme precursors inhibit the activity of the mature portions, while some of the prosequences have the function to help folding of the mature portions, such as subtilisin E of Bacillus subtilis, carboxypeptidase Y of S. cerevisiae, and lipases from Rhizopus sp. [24, 25]. The lipase secreted from R. oryzae, similar to the lipases from R. chinensis, Rhizomucor miehei and Fusarium heterosporum, is synthesized as a precursor form with a presequence (23 amino acid residues) and a prosequence (97 amino acid residues) at the N-terminal side of the mature portion (268 amino acid residues) [24, 25]. In E. coli, the activity of the prolipase could reach 100 U/mL, while the mature portion of ROL was expressed as an insoluble form without activity. The mutation studies demonstrated that the prosequence of ROL seems to facilitate the folding by providing an intramolecular thiol-disulfide reagent, and proROL is also significantly more stable against thermal inactivation than mROL [25, 26]. For the expression of R. arrhizus lipases in P. pastoris, the pro-form lipase (r28RAL) and the mature portion of the lipase in the supernatant reached 91 mg/L and 80 mg/L, respectively . Takahashi et al.  explored the role of the prosequence of ROL expressed in S. cerevisiae and indicated that the prosequence might support the correct folding of its mature portion and its subsequent secretion from the yeast cells.
In this study, we constructed a chimeric lipase from R. oryzae by replacing the prosequence with that from R. chinensis lipase and successfully expressed in P. pastoris at high-level. The chimera was characterized and its performance for biodiesel production from non-edible tung oil was investigated by response surface methodology.