3.5 Functional analysis of candidate genes in HupA...
Gene deletion was conducted to analyze the association between the candidate genes and Hup A biosynthesis.LDC genes(Cg LDC1，Cg LDC2)，CAO genes(CgCAO4)，PKS genes(Cg PKS35，Cg PKS14，Cg PKS21)and epigenetic genes(CgClr4，Cg-Spt10，CgSAS-2，CgClr3，CgSir2-3，CgSir2-6)were knocked out by homologous deletion.All genes were deleted successfully except CgSpt10 and CgSir2-3.
To eliminate the compensation effect，we not only knocked-out Cg LDC1 and Cg LDC2 individually，but also knocked-out both at one time.However，we found that Hup A production of the single deletion mutants of LDC was not changed，and that of double deletion of LDC genes increased by 83%(Figs.9).For the CAO and PKS genes，there was no differential Hup A production in the single deletion mutant fermentation broth(Figure 9).
The Hup A production of deletion mutant strainsΔCgClr4，ΔCgClr3，andΔCgSir 2-6 were reduced by 13.46%，22.26%，and 18.37%，respectively，while the mutant strainΔCgSAS-2 was not changed(Figure 10).Retro-complementation of the CgClr4，CgClr3，and CgSir2-6 mutants restored the total Hup A production(Figure 11).
Figure 9 Comparison of HupA production from culture extracts from wild type(WT)C.gloeosporioides Cg01 and LDC，CAO，and PKSgene knock out mutants(A：LDC；B：CAO；C：PKS)
The experiment was performed in triplicate.Mean values and standard deviations are given.Asterisks above the bars denote significant differences in the measurements of the indicated strains compared to the WT.*，p＜0.05；**，p＜0.01.(https://www.daowen.com)
Figure 10 Comparison of Hup A production from culture extracts from wild type(WT)C.gloeosporioides Cg01 andΔCgClr4，ΔCgSAS-2，ΔCgClr3，andΔCgSir2～6 mutants
The experiment was performed in triplicate.Mean values and standard deviations are given.Asterisks above the bars denote significant differences in the measurements of the indicated strains compared to the WT.*，p＜0.05；**，p＜0.01.
Figure 11 Comparison of HupA production from culture extracts from wild type(WT)C.gloeosporioides Cg01，epigenetic gene knock-out mutants，and the corresponding retro-complementation strains
The experiment was performed in triplicate.Mean values and standard deviations are given.Asterisks above the bars denote significant differences in the measurements of the indicated strains compared to the WT.*，p＜0.05；**，p＜0.01.