Further Enhancement Of Individual Ganoderic Acid Production By Integrating Vitreoscilla Hemoglobin Gene Overexpression And Calcium Ion Addition In Static Liquid Culture Of Ganoderma Lingzhi

Ganoderic acids(GAs),a kind of highly oxygenated C30 lanostane-type triterpenoids produced by Ganoderma lingzhi,exhibit a variety of pharmacological activities such as anti-HIV and antitumor.These GAs have two types of carbon skeletons.Type ⅠGA only have one double bond in the lanostane-skeleton,while type ⅡGA possess two conjugated double bonds.The mycelia fermentation of G.lingzhi is considered as a promising approach for the efficient production of GA.The low yield of GA in the mycelia fermentation of G.lingzhi inhibits GA clinical study and commercial application,therefore it is necessary to further improve GA production in mycelia fermentation of G.lingzhi.In this work,an integrated approach by combining cultivation strategies and genetic manipulation was developed to further enhance GA production in mycelia fermentation of G.lingzhi.The effect of the Vitreoscilla hemoglobin(VHb)expression and calcium ion addition strategies on GA biosynthesis were analyzed in static liquid cultivation of G.lingzhi.The results showed that the maximum contents of individual GA-O,GA-Mk,GA-T,GA-S and GA-Me under the integrated approach were 1451.33 ± 67.50,179.66 ± 5.06,1320.59 ± 20.84,1431.23 ± 79.74,and 1283.81 ± 85.13?g/ 100 mg cell dry weight(CDW),respectively,which were 2.66-,2.55-,2.52-,3.50-,5.98–folds higher than those in static liquid culture of the wild-type strain(the control).Especially,the maximum contents of the GA-O,GA-S,and GA-Me under the integrated approach have 16.67%,22.54% and 40.88% increasement than the highest record as ever published,respectively.These results showed that the VHb expression can be combined with calcium ion addition to promote the GA production more effectively in G.lingzhi.To well understand why the GA production was significantly improved in G.lingzhi under the integrated approach,we measured the accumulation of intermediates(squalene and lanosterol)and the expression of four important GA biosynthetic genes,3-hydroxy-3-methylglutaryl coenzyme A reductase(HMGR),farnesyl-diphosphate synthase(FPS),squalene synthase(SQS)and the cytochrome P450 monooxygenase CYP-5150L8 in different cultivation strategies.The maximum accumulations of squalene and lanosterol were 1.50±0.28 ?g/ g and 106.55±2.74 ?g/ g CDW under the integrated approach on day 9,respectively,which were 2.0-and 1.42-times higher than those in the control.The maximum transcription levels of hmgr,fps,sqs and cyp-5150l8 were up-regulated by 2.56-,3.31-,2.59-and 6.12-fold under the integrated approach,respectively.These results suggest that the enhanced GA production under the integrated approach may be attributed to the increased GA biosynthesis precursors supply and the up-regulated expression levels of GA biosynthetic genes.Previous reports show that GA distribution can be altered by environmental factors in liquid culture of G.lucidum.However,the effect of genetic manipulation of G.lingzh on GA distribution has not been reported yet.In this work,we analyzed the effect of VHb expression on the ratio of type Ⅰ: type Ⅱ GA in static liquid culture of G.lingzhi.The ratio of typeⅠ: type ⅡGA were 0.422 and 0.432 on day 12 and 15 in the VHb-expressing strain,respectively,which have 12.53% and 20.67% increasement compared with those in the wild-type strain.The results showed that VHb expression can increase the ratio of type Ⅰ: type ⅡGA.The increased ratio of type Ⅰ: type ⅡGA may be due to stimulated activities of oxygenase involved in the GA biosynthesis pathway when the VHb expressed in G.lingzhi.The cytochrome P450 monooxygenases CYP512A2(A2),CYP512V2(V2)and CYP512A13(A13)might be involved in the oxidative modification of the lanostane-skeleton to form GAs in G.lingzhi,thus the transcription levels of a2,v2 and a13 were exaimed by qRT-PCR in static liquid culture of the wild-type and the VHb-expressing G.lingzhi.Results showed that the VHb expression up-regulated the transcription levels of a2,v2 and a13 by 2.28-,2.65-and 3.54-fold,respectively.Our results suggest that the improved ratio of typeⅠ: type Ⅱ GA may be related to the up-regulation of a2,v2 and a13 in G.lingzhi.The detail roles of these P450 s in regulating GA heterogeneity need to be further investigated in futhre.Our results showed that integrating the Vitreoscilla hemoglobin(VHb)expression and calcium ion addition strategy can more effectively improve GA production in static liquid cultivation of G.lingzhi.In addition,the ratio of typeⅠ: type Ⅱ GA was increased in static liquid culture of the VHb-expressing G.lingzhi.This work is useful for large-scale production of GA and further understanding the regulation mechanism of GA heterogeneity in G.lingzhi.