## 4 Discussion  
LiP was the first lignin degradation enzyme identified within the extracellular enzymes secreted by _P. chrysosporium_. It is the key functional enzyme for lignin degradation and has great commercial value and broad application prospects. However, the LiP produced by fungi in nature has shown some disadvantages, such as high nutritional requirements, low yields and low enzyme activity, which to a great extent hinders the application of LiP for industrial purposes. The heterologous expression of LiP has previously been explored using various hosts. In particular, the heterologous expression of LiP in baculovirus expression systems has been reported but with low enzyme activity detected in the growth medium [31]. In an _Escherichia coli_ expression system, _P. chrysosporium_ LiP was expressed as inactive inclusion bodies, although subsequent activation was obtained in vitro [32; 33]. LiP was also expressed in an _Aspergillus niger_ expression system, but extracellular LiP activity produced from this expression system was weak [34]. Meanwhile, LiP expressed in the yeast _Pichia_ expression system [19; 30] has also been reported, but _Pichia_ is not a food-grade yeast and induction of expression from a foreign gene expression is driven by methanol, which is not acceptable by food and feed industries. _C. jadini_ is a promising industrial strain and has been certified as food-grade yeast by the FDA. It can be directly used as a safe single-cell protein in feed additives and can also be directly used in the production of enzyme preparations. The fermentation broth does not need to be separated and purified, which is time-saving and economical. Therefore, genetic modification of _C. jadini_ provides the strain with broad application prospects. For example, in the silage industry, the use of safe food-grade strains (such as _C. jadini_) as expression systems for improvement and application is particularly exciting. The processing of silage relies on a carbon source metabolic pathway; therefore, the _C. jadini_ expression system is an attractive option to express the lignin-degrading enzyme, lignin peroxidase. Figure 8: Phenotype profiles of the ZHMX4 and _C. jadini_ as determined using the Biolog System. ZHMX4 (upper panel; left—graphic representation, right—numerical values) and _C. jadini_ (lower panel; left—graphic representation, right—numerical values). Plates were read after 24, 48 and 72 h of incubation. Only 72 h data are shown here. A1 is used as the reference for the rows A–C, and D1 is used as the reference for the rows D–H. In this study, the _lip_ gene of white-rot fungus (_P. chrysosporium_) was cloned, and heterologously expressed in the food-grade yeast, _C. jadinii_, for the first time. The recombinant strain ZHMX4 expressing LiP was constructed, and its genetic stability and enzyme activity were evaluated. The prokaryotic DNA sequence from the homologous integrated expression vector was deleted before transformation into _C. jadini_. All the genetic elements of the homologous integrated DNA were taken from the probiotic _C. jadini_ and the non-toxic and harmless fungi _P. chrysosporium_. There were no antibiotic resistance genes; no drift, diffusion and integration of resistance genes and no other toxic protein genes to eliminate any potential harm to the environment or human and animal safety. This would enable the product to reach the food-grade level and be added directly to food and feed. ZHMX4 still retained the inserted gene after 100 generations, indicating that the foreign gene was preserved and maintained high genetic stability even after multiple rounds of subculturing. The enzyme activity assay also confirmed that the _lip_ gene was inserted into the chromosome of _C. jadini_ and could express active LiP. Phenotypic analysis showed obvious changes in the metabolic characteristics of ZHMX4 compared to _C. jadini_. Thus, ZHMX4 is a food-grade genetically engineered strain with great potential for lignin degradation.