Enhanced production of individual ganoderic acids by integrating Vitreoscilla haemoglobin expression and calcium ion induction in liquid static cultures of Ganoderma lingzhi

Summary Ganoderic acids produced by Ganoderma exhibit anticancer and antimetastatic activities. A novel approach by combining Vitreoscilla haemoglobin (VHb) expression and calcium ion induction was developed to enhance ganoderic acid (GA) production in liquid static cultures of G. lingzhi. The maximum contents of GA‐O, GA‐S and GA‐Me were 1451.33 ± 67.50, 1431.23 ± 79.74 and 1283.81 ± 85.13 μg per 100 mg cell weight, respectively under the integrated approach, which are the highest contents as ever reported in Ganoderma. The contents of squalene and lanosterol were increased by 2.0‐ and 3.0‐fold in this case compared with those in the control. The transcription levels of 3‐hydroxy‐3‐methylglutaryl coenzyme A reductase, farnesyl‐diphosphate synthase, squalene synthase and cytochrome P450 CYP5150L8 were upregulated by 2.56‐, 3.31‐, 2.59‐ and 6.12‐fold respectively. Additionally, the expression of VHb improved the ratio of type I to type II GA in liquid static cultivation of G. lingzhi. The transcription levels of cyp512a2, cyp512v2 and cyp512a13, candidate cytochrome P450 genes involved in oxidative modification of the lanostane skeleton in GA biosynthesis, were also increased by 2.28‐, 2.65‐ and 3.54‐fold in the VHb‐expressing strain respectively. Our results illustrated that the approach described here efficiently improved GA production in G. lingzhi fermentation.


Introduction
Ganoderma lingzhi has been used to prevent and treat various human diseases for thousands of years in East Asia (Paterson, 2006). Ganoderic acids (GAs), highly oxygenated C30 lanostane-type triterpenoids produced by Ganoderma species, have a variety of important pharmacological activities. For instance, Ganoderic acid (GA)-Mk and GA-S induce the apoptosis of HeLa cells (Liu and Zhong, 2011;Liu et al., 2012). GA-T and GA-Me inhibit lung cancer cell growth and metastasis (Tang et al., 2006;Wang et al., 2007), and GA-O has cytotoxicity against lung and HeLa cancer cells . GAs have two types of carbon skeletons ( Fig. 1): GA-Mk, GA-S, GA-T and GA-Me (type II) possess two conjugated double bonds on the tetracyclic rings, while GA-O (type I) has only one double bond on the lanostane skeleton and more oxidative modification at C-3, C-7, C-15 and C-22 .
Ganoderic acids are synthesized via the mevalonate/ isoprenoid (MVA) pathway in Ganoderma species (Xu et al., 2010b). Lanosterol, an important intermediate, is converted to different individual GAs through a series of modifications, including oxidation, reduction and acylation reactions, in Ganoderma lucidum (Xu et al., 2010b;Chen et al., 2012;Zhang et al., 2017a,b). Some key genes involved in the upstream cyclization pathway of the lanostane skeleton, including 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR), farnesyl-diphosphate synthase (FPS) and squalene synthase (SQS), have been cloned from G. lucidum Zhou et al., 2014). Recently, the cytochrome P450 monooxygenase CYP5150L8 gene, which catalyses the three-step oxidation of lanosterol at C-26 in GA biosynthesis, was also been isolated and characterized . Other cytochrome P450 genes, such as cyp512a2, cyp512v2 and cyp512a13, might be involved in the oxidative modification of the lanostane skeleton to form different individual GAs in Ganoderma (Chen et al., 2012;Jiang et al., 2018;Yang et al., 2018).
Due to their important pharmacological activities, many strategies have been used to increase the production of GAs during mycelia fermentation. Progress has been achieved in the last several decades in enhancing GA production in G. lucidum by the manipulation of fermentation strategies (Xu et al., 2010a;Upadhyay et al., 2014;Wang et al., 2016), addition of metal ions and elicitors You et al., 2013You et al., , 2017Zhang et al., 2014;Gu et al., 2018) and genetic engineering Xu and Zhong, 2015;Li et al., 2016a,b,c;Zhang et al., 2017a,b). However, further improvement of GA production is necessary to meet the demand of increasing preclinical studies and commercial applications.
The combination of both a good producer strain and an efficient bioprocess strategy is a promising approach for the enhancement of secondary metabolites. For example, the integration of precursor feeding and tryptophan decarboxylase gene overexpression leads to a higher accumulation of indole alkaloids than either strategy alone in Catharanthus roseus (Canel et al., 1998;Whitmer et al., 2002). In Salvia miltiorrhiza, it is more effective to combine elicitor treatments with transgenic technology for high enhancement of tanshinone content (Hao et al., 2015). However, the effect of integrating genetic modification and fermentation strategies in the production of secondary metabolites has not been studied in Ganoderma. A liquid static cultivation strategy is effective in promoting the production of GAs by G. lucidum compared with liquid shaking culture (Xu et al., 2010a). Xu et al. reported that calcium ion addition improved GA production in a liquid static culture of G. lucidum . Our recent work found that the heterologous expression of the Vitreoscilla haemoglobin (VHb) gene increased GA biosynthesis in submerged cultivation of G. lucidum (Li et al., 2016a). Therefore, in this study, we observed the effects of VHb expression and calcium ion induction on GA biosynthesis in a liquid static culture of G. lingzhi.
Many secondary metabolites have similar chemical structures but are bioactively different (Zhong and Yue, 2005). Therefore, the distribution of secondary metabolites is of great interest for biotechnological applications. External environmental factors and genetic manipulation usually play important roles in regulating the heterogeneity of secondary metabolites. For instance, the ratio of the protopanaxatriol-type to the protopanaxadiol-type of ginsenosides can be altered by changing oxygen pressure and with the addition of phenobarbital in cell cultures of Panax notoginseng (Han and Zhong, 2003;Yue and Zhong, 2008). In Aurantiochytrium sp., heterologous expression of the VHb gene increases the ratio of astaxanthin to beta-carotene significantly (Suen et al., 2014). In G. lucidum, Zhang et al. reported that enriching oxygen level affects the individual GA distribution in a liquid static culture (Zhang and Zhong, 2010). Liang et al. found that phenobarbital altered the individual GA distribution in a liquid static culture of G. lucidum (Liang et al., 2010). However, the effect of genetic modification of Ganoderma on individual GA distributions has not been investigated yet. In this work, an integrated approach by combining VHb expression and calcium ion induction was applied to enhance the production of individual GAs in a liquid static culture of G. lingzhi. Moreover, the effect of heterologous expression of the VHb gene on individual GA distributions was also analysed in G. lingzhi.

Results and discussion
VHb expression enhanced the production of individual GAs in liquid static cultures of G. lingzhi The kinetics of cell growth of the wild-type (WT) and VHb-expressing G. lingzhi (VHb) in liquid static culture conditions are shown in Fig 2A. No significant difference was observed between WT and VHb in the liquid static culture mode. This may be due to the sufficient supply of oxygen and a relatively low cell density in the WT and VHb-expressing strain under the normal culture condition (Li et al., 2016a). 13 lg per 100 mg CDW, respectively, which were increases of 32%, 56%, 24%, 66% and 48% compared with VHb alone. In particular, the contents of GA-O, GA-S and GA-Me obtained under the integrated approach were much higher than previously reported highest levels, including those using static liquid of the WT , phenobarbital induction (Liang et al., 2010) and higher oxygen levels (Zhang and Zhong, 2010). These results suggest that combining genetic engineering and the fermentation strategy can more efficiently improve the production of individual GAs in G. lingzhi. Similar results have also been reported for Streptomyces graminearus, in which combined gene cluster engineering and glycine feeding increase gougerotin production by 2.5-, 1.25-,and 1.3fold higher compared to the WT strain, the engineered strain and the WT strain by glycine feeding respectively (Jiang et al., 2013). Exogenous feeding of nutrients to cultures of the engineered S. tsukubaensis leads to 3.2-, 1.3-and 2.6-fold improvement in FK506 production compared with those in the WT strain, the engineered strain and the WT strain fed with nutrients respectively (Huang et al., 2013). Table 1 shows that the maximum squalene content with the integrated approach was 1.50 AE 0.28 lg g CDW À1 on day 9, which was 2.0-fold higher than that under control conditions. Although the content gradually decreased to 0.50 AE 0.28 lg g CDW À1 on day 12, it still maintained a higher value than the control. The concentration of lanosterol with the integrated approach reached a maximum of 106.55 AE 2.74 lg g CDW À1 on day 9, but declined to 81.74 AE 1.80 lg g CDW À1 on day 12. The maximum level of lanosterol with the integrated approach was 1.4 times higher than that under the control conditions. Fig. 3A shows that the maximum transcription levels of hmg, fps, sqs and cyp-5150 l8 observed on day 9 under the integrated approach were 2.56, 2.59, 3.31 and 6.12 times higher than those in the control conditions respectively. These values decreased from day 9 to day 12, but still maintained higher levels than those in the control.

Effects of the integrated approach on the accumulation of intermediates and on GA biosynthetic gene expression
The contents of lanosterol and squalene and the expression of GA biosynthetic genes were higher under the integrated approach compared to the control liquid Table 1. Contents of squalene and lanosterol in liquid static cultures of wild-type G. lingzhi (the control) and VHb-expressing strain with calcium ion induction (the integrated approach).

Culture conditions
Cultivation time Biomass (g l À1 ) Squalene content (lg g DCW À1 ) Lanosterol content (lg g DCW À1 ) The static culture of G. lingzhi. These trends were positively correlated with the enhanced production of individual GAs, which indicated that that the improved GA production could be attributed to increased precursor supply and the upregulated expression of biosynthetic genes. In Antrodia cinnamonea and Paraconiothyrium, a higher supply of precursors increases the production of triterpenoids and diterpene respectively (Chen et al., 2016;Solima et al., 2017). Previous reports have also found that there is a positive correlation between the expression levels of triterpene biosynthetic genes and the contents of ginsenoside and astragalosides (Jiao et al., 2016;Li et al., 2016b). This was also observed in our previous works with squalene synthase overexpression and the integration of nitrogen limitation and calcium ion addition (Zhou et al., 2014;Li et al., 2016c).
VHb expression increased the ratio of type I to type II GAs in static liquid cultures of G. lingzhi As shown in Table 2, the ratios of type I to type II GAs were 0.422 and 0.432 in the VHb strain, while the ratios were 0.375 and 0.358 in the WT of G. lingzhi on day 12 and day 15 respectively. VHb expression thus increased the ratio of type I to type II GAs in static liquid cultures of G. lingzhi. Fig. 3B shows that the transcription levels of cyp-512a2 and cyp-512v2 in the VHb strain were 2.28-and 2.65-fold higher than those in the WT on day 9. For cyp-512a13, the transcription level in the VHb strain was 3.54-fold higher than that in the WT on day 6. The transcription levels of these P450 genes were thus significantly induced by VHb compared to the WT. Oxidative modification of the lanostane skeleton plays an important role in the biosynthesis of GA. The increased ratio of type I to type II GAs may be due to the stimulated activities of oxygenase involved in the GA biosynthesis pathway (Stark et al., 2015;Li et al., 2016a). The transcription levels of cyp512a2, cyp512v2 and cyp512a13 were upregulated in the VHb strain, suggesting that the increased ratio may be related to higher transcription levels of those cytochrome P450 genes. In P. notoginseng, the induced activity of the cytochrome P450 enzyme protopanaxadiol 6-hydroxylase increases the ratio of protopanaxatriol-type to protopanaxadiol-type ginsenoside (Yue and Zhong, 2008). The detailed roles of these cytochrome P450s in regulating GA distribution need to be further investigated in the future.  In summary, we demonstrated that an integrated approach that combines VHb expression and calcium ion induction can effectively improve the production of individual GAs in liquid static cultivation of G. lingzhi. In addition, the expression of the VHb gene increased the ratio of type I to type II GAs in liquid static cultures of G. lingzhi. This work will be useful for large-scale production of individual GAs and to understand the regulatory mechanisms of GA heterogeneity in G. lingzhi.

Experimental procedures
Strains and culture conditions Ganoderma lucidum CGMCC 5.616 was bought from the China General Microbiological Fermentation Center and is conspecific with Ganoderma lingzhi (Zhang et al., 2017a,b). The VHb-expressing G. lingzhi was constructed in a previous study (Li et al., 2016a). The pre-culture and fermentation details of the liquid static culture of G. lingzhi were described before (Xu et al., 2010a) and are shown in the Appendix S1. CaCl 2 was dissolved in water and was sterilized by filtering through 0.22-lm polyvinylidene difluoride syringe filters (Millipore, Germany) before their additions into cultures. 10 mM of CaCl 2 was added when shaking flask cultures were converted to liquid static cultures in 10-cm-diameter plate for incubation.
Sampling, and measurements of cell dry weight, individual GAs, squalene and lanosterol For sampling, three plates were taken each time. Cell dry weight was measured by a gravimetric method. Individual GAs, squalene and lanosterol were extracted and determined by methods described earlier Zhou et al., 2014) (see also Appendix S1).

Total RNA extraction and cDNA synthesis
The total RNA from G. lingzhi was extracted with TRIzol Reagent (Invitrogen, Carlsbad, CA, USA) and treated with DNase I to remove residual DNA. One microgram of total RNA was reverse-transcribed by using the Prime-Script TM RT reagent Kit (TaKaRa, Dalian, China).
Isolation of cytochrome P450 genes from G. lingzhi Partial cDNAs of cytochrome P450 monooxygenases CYP-5150L8, CYP512 A2, CYP512 V2 and CYP512 A13 were isolated by PCR amplification using primers shown in Table S1. The amplified fragments were ligated into the pMD 19-T Simple Vector by the T4 DNA ligase system (TaKaRa) and sequenced using both M13 + and M13À as primers (Sangon, Shanghai, China).

Real-time quantitative PCR (qRT-PCR) analysis
The transcription levels of hmgr, fps, sqs, cyp-5150 l8, cyp-512a2, cyp-512v2 and cyp-512a13 were measured by qRT-PCR. The qRT-PCR primer sequences of the 18S rRNA gene, hmgr, fps and sqs are described elsewhere (Xu et al., 2010a;Zhao et al., 2011). The qRT-PCR primers for cyp-5150 l8, cyp-512a2, cyp-512v2 and cyp-512a13 are listed in Table S1. Real-time PCR was performed using SYBR â Premix Ex Taq TM II (TaKaRa) according to the manufacturer's instructions. The cDNA was quantified using a CFX96 TM real-time PCR detection system (Bio-Rad, Inc., Hercules, CA, USA). The transcription levels of the target genes were normalized with that of the G. lingzhi 18S rRNA gene. A transcription level of 1 was defined for samples from the liquid static culture of the wild-type strain, and the transcription levels in other cultivation strategies were expressed as fold changes of the mRNA levels over the reference level. Relative gene transcription levels were analysed according to the 2 ÀDDC T method.

Statistical analysis
All of the data were generated from three independent samples. Data are expressed as average values AE standard deviation. Statistical analysis was performed using Student's paired t-test. The difference was considered significant when P < 0.05 in a two-tailed analysis.