Enhancing production of ergosterol in Pichia pastoris GS115 by over-expression of 3-hydroxy-3-methylglutaryl CoA reductase from Glycyrrhiza uralensis.

The rate-limiting enzyme in the mevalonic acid (MVA) pathway which can lead to triterpenoid saponin glycyrrhizic acid (GA) is 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR). In order to reveal the effect of copy number variation in the HMGR gene on the MVA pathway, the HMGR gene from Glycyrrhiza uralensis Fisch. (GuHMGR) was cloned and over-expressed in Pichia pastoris GS115. Six recombinant P. pastoris strains containing different copy numbers of the GuHMGR gene were obtained and the content of ergosterol was analyzed by HPLC. The results showed that all the recombinant P. pastoris strains contained more ergosterol than the negative control and the strains with 8 and 44 copies contained significantly more ergosterol than the other strains. However, as the copy number increased, the content of ergosterol showed an increasing-decreasing-increasing pattern. This study provides a rationale for increasing the content of GA through over-expressing the GuHMGR gene in cultivars of G. uralensis.

Introduction

Besides the use as an industrial raw material and tobacco additive, the roots of Glycyrrhiza uralensis Fisch. are widely used in many Chinese herbal remedies for their ability to nourish “Qi”, alleviate pain, tonify the spleen and stomach, eliminate phlegm and relieve cough. The source of this pharmacological activity is a number of active components of which glycyrrhizic acid (GA) is considered the most important. This has led to its adoption as a marker compound of the quality of G. uralensis. Many studies have shown that GA possesses antiinflammatory, antiturmor and immune-stimulating activities.

Excessive exploitation of wild G. uralensis plants in the years leading up to 2000 decreased the supply to such an extent that the Chinese government imposed restrictions on their collection. As a result, cultivars have now become the main source of this herb. However, the low content of GA in these cultivars has placed severe restrictions on their sustainable development. Attempts have been made to solve this problem using cell suspensions of G. uralensis but without success. We therefore decided to genetically engineer G. uralensis plants through modifying the triterpene biosynthetic pathway which leads to the formation of GA.

In the biosynthesis of GA, the rate-limiting enzyme is 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR) which catalyzes the reaction of HMG-CoA and NADPH to form mevalonic acid (MVA). Many previous studies have shown that the accumulation of terpenes is significantly increased by increasing the content of the HMGR gene but, to date, over-expression of the HMGR gene in G. uralensis (GuHMGR) to increase the production of GA has not been reported. In this study, we investigated how copy number variation (CNV) of the GuHMGR gene affects the formation of ergosterol. We maintain that the results indicate that over-expression of the GuHMGR gene increases the accumulation of GA in cultivars of G. uralensis.

Materials and methods

Construction of the yeast expression vector containing GuHMGR gene

NotI and SnaBI of pPIC9K (Fig. 1) were selected as the specific enzyme cutting sites to insert the GuHMGR gene. Primer pairs with the specific enzyme sites underlined are as follows:

Figure 1

Structure of pPIC9K.

HF: 5′-CGGTACGTAATGGACGTTCGCCGGAG-3′ (SnaBI)

HR: 5′-ATAGCGGCCGCTGGAGGCTTTCGTTATTGGT-3′ (NotI)

The cycling parameters of PCR were as follows: 94 °C for 5 min; 30 cycles of 94 °C for 30 s, annealing at 64 °C for 30 s, extension at 72 °C for 2 min; and a final extension at 72 °C for 10 min. The amplified fragments were purified and subcloned into pMD19-T (Takara, Japan). The resulting vector (GuHMGR-T) was digested with SnaBI (2 h at 37 °C) and NotI (2 h at 37 °C) and then subcloned into pPIC9K (Invitrogen, USA). The resulting recombinant pPIC9K–GuHMGR plasmid was transferred into the disarmed E. coli DH5α and sequenced for correct insertion.

Construction of recombinant P. pastoris containing GuHMGR gene

The recombinant pPIC9K–GuHMGR plasmid was linearized by restriction enzyme SalI and mobilized by electroporation (1500 V, 25 µF, 400 Ω) into the disarmed P. pastoris GS115 (Invitrogen, USA). An aliquot (0.5 mL) of yeast peptone dextrose (YPD) medium was then added and the cells were cultured at 30 °C, 200 rpm for 1 h. An aliquot (200 µL) of the suspension was placed on minimal dextrose (MD) solid medium and cultured at 30 °C for 2 days. Single colonies were removed and incubated on minimal medium (MM) and MD solid medium simultaneously at 30 °C for 2–4 days; the colonies growing on both MM and MD media were selected.

PCR was used to check that the recombinant P. pastoris contained the GuHMGR gene. The single colonies were used as PCR template and primers were as follows: forward primer, 5′-TACTATTGCCAGCATTGCTGC-3′; reverse primer, 5′-GCAAATGGCATTCTGACATCC-3′. The cycling parameters were as follows: 94 °C for 5 min; 30 cycles of 94 °C for 30 s, annealing at 60 °C for 30 s, extension at 72 °C for 2 min; and a final extension at 72 °C for 10 min.

Selected recombinant P. pastoris was induced to express the GuHMGR gene using BMGY and BMMY liquid media (30 °C, 250 rpm). The supernatant from a 96 h culture was examined by 12% SDS-PAGE using Coomassie brilliant blue staining. P. pastoris containing a void vector was used as a negative control.

Copy number determination

The GAP gene was selected as the internal control gene for real-time PCR. The primer pair of GAP (GenBank accession number: U62648) was as follows: GF: 5′-CACAATGGCTATCACTGTCG-3′; GR: 5′-GACACACTACAGCCCGCATT-3′. The primer pair of the GuHMGR gene was as previously stated. The cycling parameters were as follows: 94 °C for 5 min; 30 cycles of 94 °C for 30 s, annealing at 60 °C for 30 s, extension at 72 °C for 2 min; and a final extension at 72 °C for 10 min. The amplified fragments were subcloned into pMD19-T and transformed into disarmed E. coli DH5α. Then the standard plasmids pMD19-T–GuHMGR and pMD19-T–GAP were obtained, extracted and diluted to 109, 108, 107, 106, 105, 104, 103, and 102 copy numbers/2 µL. For real-time PCR analysis, the primer pairs in Table 1 were used with the following cycling parameters: 95 °C for 10 min; 40 cycles of 95 °C for 15 s, 60 °C for 60 s, saving at 4 °C. Standard curves of Ct (Cycle threshold) on the X-axis and log (concentration of standard plasmid) on the Y-axis were constructed. All recombinant P. pastoris strains were amplified by real-time PCR. The ratio of the Y values of GuHMGR and GAP was taken as the copy number of the GuHMGR gene in each recombinant P. pastoris strain.

Table 1

Primers used for real-time PCR analysis.

Gene	Primer
GAP	rGF: 5′-GGTATTAACGGTTTCGGACGTATTG-3′
rGR: 5′-GATGGTGACAGGGTCTCTCTCTTGG-3′
GuHMGR	rHF: 5′-CACGGTTTCCTCGTCTTCAA-3′
rHR: 5′-CGTCTACCTCCTCGGCTTCTT-3′

Semi-quantitative RT-PCR analysis

Total RNA was isolated from different recombinant P. pastoris strains using a yeast RNA rapid extraction kit (Beijing BoMaiDe Medical Technology Co., Ltd.). To remove plasmid DNA, RNase-free DNase I enzyme (Tiangen Biotech Co., Ltd.) was used according to the manufacturer׳s instructions. Spectrophotometry was used to determine the concentration of RNA. The cycle number was set at 18, 20, 22, 24 and 26, and the optimal cycle number determined by electrophoresis in 1% (w/v) agarose gel. The cycling parameters of RT-PCR were as follows: 50 °C for 30 min; 94 °C for 2 min; optimal cycles of 94 °C for 30 s, annealing at 60 °C for 30 s, extension at 72 °C for 45 s; and a final extension at 72 °C for 10 min; saving at 4 °C. The primer pair, GF (5′-CACAATGGCTATCACTGTCG-3′) and GR (5′-GACACACTACAGCCCGCATT-3′), was used to amplify the GAP gene as an internal control.

Assay of ergosterol in recombinant P. pastoris

HPLC analysis of ergosterol in samples was carried out on a Waters 2695 system equipped with a Phenomenex LUNA C18 column (250 mm×4.6 mm, 5 µm) using a mobile phase of methanol:water 97:3 (v/v) delivered at a flow rate of 1.0 mL/min. The detection wavelength was 283 nm and the injection volume 20 µL.

A stock solution of ergosterol (purity: 97.7%) containing 11.38 mg in 10 mL absolute ethanol was used to prepare a series of standard solutions containing 0.01%, 0.05%, 0.1%, 0.5%, 2%, and 5% of the stock solution in absolute ethanol. Intra-day precision (as relative standard deviation, RSD) was determined by replicate analysis (n=6) of a solution containing 5.559 µg/mL ergosterol. The limit of detection (LOD) and lower limit of quantity (LLOQ) were determined using sequentially more dilute solutions of ergosterol. Recovery was assessed using 9 samples of blank P. pastoris cells accurately weighed (50 mg) and spiked with 2.91 µg, 5.05 µg and 8.41 µg ergosterol.

Recombinant P. pastoris strains containing different copy numbers of the GuHMGR gene were induced to express the gene; P. pastoris GS115 without the GuHMGR gene was used as negative control. All 96 h cultures were collected by centrifugation at 5000 rpm and lyophilized. Samples of the dried powders (50 mg) were extracted into 8 mL ethyl acetate by ultrasonication for 1 h. The ethyl acetate was evaporated to dryness, the residue reconstituted in 2 mL ethanol analyzed for ergosterol.

Results

Construction of the yeast expression vector containing the GuHMGR gene

A 1745 bp fragment was shown by PCR and BLAST analysis to have a 99% identical sequence to that of the GuHMGR gene (GenBank accession number: GQ345405.1). It was successfully inserted at the SnaBI–NotI site of pPIC9K to give the recombinant plasmid pPIC–GuHMGR shown in Fig. 2 where lane 1 is the marker and lanes 2 and 3 are fragments obtained by PCR with the correct length.

Figure 2

PCR analysis of the construction of recombinant plasmid pPIC–GuHMGR. Lane 1: marker; lanes 2 and 3: fragments obtained by PCR.

The linearized pPIC–GuHMGR was transformed to P. pastoris GS115. Most single colonies of recombinant P. pastoris simultaneously growing on MM and MD media were shown by PCR to have the correct fragment length. After inducing for 96 h, the negative control was dark yellow while the recombinant P. pastoris was yellowish-white. SDS-PAGE (Fig. 3) showed a band between 86 and 47 kDa in samples from recombinant P. pastoris which was not present in the negative control. These results demonstrate that the construction and inducible expression of recombinant P. pastoris strains containing the GuHMGR gene were successful.

Figure 3

SDS-PAGE analysis of the expression of the GuHMGR gene. Lane 1: marker; lanes 2–4: recombinant P. pastoris containing the GuHMGR gene; lane 5: negative control.

Copy number determination of the GuHMGR gene in transgenic P. pastoris

PCR and sequencing showed that the standard plasmids were correct. The melting curves of the GAP and GuHMGR genes were both unimodal suggesting that the primers used in real time PCR were specific. Using real time PCR, two fragments with 220 and 237 bp were obtained which sequencing and BLAST analysis showed were the GuHMGR gene and the GAP gene of P. pastoris, respectively. Their standard curves were described by the equations Y=−2.609X+32.21 (R2=0.995) and Y=−2.994X+35.27 (R2=0.997), correspondingly. The copy numbers of the GuHMGR gene in the different transgenic P. pastoris strains were found to be 1, 2, 4, 8, 13 and 44 (Table 2).

Table 2

Copy numbers of the GuHMGR gene in different recombinant P. pastoris.

No. of strain	Average Ct value		Y value in standard curve		Copy number of GuHMGR (GuHMGR/GAP)
GAP	GuHMGR	GAP	GuHMGR
1	23.10	21.18	1.2×104	1.7×104	1
2	17.29	15.51	1.0×106	2.5×106	2
3	24.26	20.83	4.7×103	2.3×104	4
4	20.38	16.81	9.4×104	8.0×105	8
5	22.36	18.06	2.0×104	2.7×105	13
6	25.12	19.07	2.5×103	1.1×105	44

Semi-quantitative RT-PCR analysis of transgenic P. pastoris

Semi-quantitative RT-PCR was employed to detect the relative abundance of the GuHMGR gene in the transgenic P. pastoris strains. The cycle number was finally fixed at 20 based on a preliminary experiment. RT-PCR analysis revealed the GuHMGR gene was expressed in all transgenic P. pastoris strains at different levels (Fig. 4b) whereas expression of the GAP gene was roughly similar (Fig. 4a). As shown in Fig. 4c, the relative expression of the GuHMGR gene in the recombinant P. pastoris strain containing 4 copies was higher than in the other strains consistent with the results listed in Table 3 (P<0.05 for the strain containing 4 copies versus all other strains).

Figure 4

Semi-quantitative RT-PCR analysis of the expression of the GuHMGR gene in different recombinant P. pastoris strains. (a) RT-PCR results of the GAP gene; (b) RT-PCR results of the GuHMGR gene (the numbers are the copy numbers of the GuHMGR gene in the recombinant P. pastoris strains); (c) relative expression of the GuHMGR gene in recombinant P. pastoris strains with different copy numbers.

Table 3

Independent t-test results of expression of the GuHMGR gene in different recombinant P. pastoris strains by RT-PCR.

Copy number	P value
1	2	4	8	13	44
1	–	0.425	0.000	0.736	0.994	0.019
2	0.425	–	0.000	0.264	0.421	0.082
4	0.000	0.000	–	0.000	0.000	0.000
8	0.736	0.264	0.000	–	0.741	0.010
13	0.994	0.421	0.000	0.741	–	0.018
44	0.019	0.082	0.000	0.010	0.018	–

The retention time of ergosterol in HPLC was 8.16 min. The standard curve was linear over the concentration range 0.1–50.0 µg/mL and described by the equation Y=3.8184×10−5X–7.9302×10−3 (R2=0.9999). The LLOQ (S/N of 10) was 2.22 ng and the LOD (S/N of 3) 0.89 ng. The assay was precise (RSD 0.0592%, n=6) with recovery in the range 97.3%–101.0% (RSD 0.55%–0.82%). The content of ergosterol in all samples is shown in Fig. 5. The results of independent t-tests of the content of ergosterol in different recombinant P. pastoris strains (n=3) are listed in Table 4. The level of ergosterol in all recombinant P. pastoris strains was 1.07–2.51 times higher than in the negative control but with increase in the copy number of GuHMGR gene; the content of ergosterol showed an increasing–decreasing–increasing pattern. For strains with copy number <4, the content of ergosterol was similar; at copy number 8, the content of ergosterol was highest (2.5 times the negative control); for copy number 13, the content of ergosterol was only 1.04 times negative control; and for copy number 44, the content of ergosterol was 1.8 times negative control. Clearly, the copy number of the GuHMGR gene influences the level of ergosterol in transgenic P. pastoris.

Figure 5

Content of ergosterol in recombinant P. pastoris strains with different copy numbers of the GuHMGR gene (n=3).

Table 4

Independent t-test results of the content of ergosterol in different recombinant P. pastoris strains (n=3).

Copy number	P value
0	1	2	4	8	13	44
0	–	0.000	0.002	0.000	0.000	0.142	0.001
1	0.000	–	0.959	0.847	0.000	0.001	0.006
2	0.002	0.959	–	0.951	0.000	0.006	0.011
4	0.000	0.847	0.951	–	0.000	0.001	0.007
8	0.000	0.000	0.000	0.000	–	0.000	0.004
13	0.142	0.001	0.006	0.001	0.000	–	0.001
44	0.001	0.006	0.011	0.007	0.004	0.001	–

Discussion

In our previous studies, we found functional genes in G. uralensis such as those of HMGR and SQS were subject to CNV. We were therefore interested to analyze the relationship between the copy number of functional genes and the content of GA acid in G. uralensis.

CNV arises from deletions, insertions, duplications, and more complex variations ranging from 1 kb to submicroscopic sizes. Genes with CNV have wide distribution, hereditability, relative stability and high heterogeneity. CNV can lead to changes in gene dosage and phenotypic character and, to date, many reports have documented a close relationship between CNV and human disease. Despite the high level of interest in CNV, there are few reports of CNV in plant genes.

In this study, recombinant P. pastoris strains containing 1, 2, 4, 8, 13 and 44 copies of the GuHMGR gene were constructed. RT-PCR analysis revealed that the GuHMGR gene was expressed in all transgenic P. pastoris strains at different levels with the strain containing 8 copies, showing highest expression as indicated by the content of ergosterol. However, with increasing copy number, the content of ergosterol did not increase in a linear fashion but showed an increasing–decreasing–increasing pattern with the strains containing 8 and 44 copies containing higher levels of ergosterol than those containing 1, 2, 4 and 13 copies.

The reason for the non-linear dependence of expression on copy number is unclear but one possibility is that it involves gene silencing caused by integration sites of exogenous genes. Recently, several studies have demonstrated feedback inhibition in over-expressing exogenous genes in plants and it may be that an increase in the GuHMGR gene results in feedback inhibition of upstream steps of the MVA pathway to reduce the accumulation of ergosterol. Then when an enzyme level decreases, feedback inhibition may be interrupted leading to reopening of the metabolic pathway which could explain why the level of ergosterol is increased in the P. pastoris strain containing 44 copies of the GuHMGR gene.

In this study, the dependence of the content of ergosterol on the copy number of the GuHMGR gene suggests that an increase in the latter could lead to an increase in the production of GA in G. uralensis. However, it must be recognized that the production of GA involves a very complex metabolic network which is regulated and controlled by many key enzymes, of which HMGR is but one. Nevertheless we maintain that the current results provide an important basis for further studies aimed at increasing the GA content of G. uralensis and exploring its biosynthesis in vitro. In addition, other herbs used in Chinese medicine such as Glycyrrhiza glabra and Glycyrrhiza inflate also produce GA and this work is relevant to further studies of its biosynthesis in these medicinal plants.