Directed evolution and expression tuning of geraniol synthase for efficient geraniol production in Escherichia coli.

To achieve an efficient production of geraniol and its derivatives in Escherichia coli, we aimed to improve the activity of geraniol synthase (GES) through a single round of mutagenesis and screening for higher substrate consumption. We isolated GES variants that outperform their parent in geraniol production. The analysis of GES variants indicated that the expression level of GES was the bottleneck for geraniol synthesis. Over-expression of the mutant GESM53 with a 5'-untranslated sequence designed for high translational efficiency, along with the additional expression of mevalonate pathway enzymes, isopentenyl pyrophosphate isomerase, and geranyl pyrophosphate synthase, yielded 300 mg/L/12 h geraniol and its derivatives (>1000 mg/L/42 h in total) in a shaking flask.

Manipulation of GES and ERG20 for geraniol overproduction in Saccharomyces cerevisiae.

Manipulation of monoterpene synthases to maximize flux towards targeted products from GPP (geranyl diphosphate) is the main challenge for heterologous monoterpene overproduction, in addition to cell toxicity from compounds themselves. In our study, by manipulation of the key enzymes geraniol synthase (GES) and farnesyl diphosphate synthase (Erg20), geraniol (a valuable acyclic monoterpene alcohol) overproduction was achieved in Saccharomyces cerevisiae with truncated 3-hydroxy-3-methylglutaryl-coenzyme reductase (tHMGR) and isopentenyl diphosphate isomerase (IDI1) overexpressed. The expressions of all above engineered genes were under the control of Gal promoter for alleviating product toxicity. Geraniol production varied from trace amount to 43.19mg/L (CrGES, GES from Catharanthus roseus) by screening of nine GESs from diverse species. Further through protein structure analysis and site-directed mutation in CrGES, it was firstly demonstrated that among the high-conserved amino acid residues located in active pocket, Y436 and D501 with strong affinity to diphosphate function group, were critical for the dephosphorylation (the core step for geraniol formation). Moreover, the truncation position of the transit peptide from the N-terminus of CrGES was found to influence protein expression and activity significantly, obtaining a titer of 191.61mg/L geraniol in strain with CrGES truncated at S43 (t3CrGES). Furthermore, directed by surface electrostatics distribution of t3CrGES and Erg20WW (Erg20F96W-N127W), co-expression of the reverse fusion of Erg20ww/t3CrGES and another copy of Erg20WW promoted the geraniol titer to 523.96mg/L at shakes flask level, due to enhancing GPP accessibility led by protein interaction of t3CrGES-Erg20WW and the free Erg20WW. Eventually, a highest reported titer of 1.68g/L geraniol in eukaryote cells was achieved in 2.0L fed-batch fermentation under carbon restriction strategy. Our research opens large opportunities for other microbial production of monoterpenes. It also sets a good reference for desired compounds overproduction in microorganisms in terms of manipulation of key enzymes by protein engineering and metabolic engineering.

Efficient diterpene production in yeast by engineering Erg20p into a geranylgeranyl diphosphate synthase.

Terpenes have numerous applications, ranging from pharmaceuticals to fragrances and biofuels. With increasing interest in producing terpenes sustainably and economically, there has been significant progress in recent years in developing methods for their production in microorganisms. In Saccharomyces cerevisiae, production of the 20-carbon diterpenes has so far proven to be significantly less efficient than production of their 15-carbon sesquiterpene counterparts. In this report, we identify the modular structure of geranylgeranyl diphosphate synthesis in yeast to be a major limitation in diterpene yields, and we engineer the yeast farnesyl diphosphate synthase Erg20p to produce geranylgeranyl diphosphate. Using a combination of protein and genetic engineering, we achieve significant improvements in the production of sclareol and several other isoprenoids, including cis-abienol, abietadiene and ß-carotene. We also report the development of yeast strains carrying the engineered Erg20p, which support efficient isoprenoid production and can be used as a dedicated chassis for diterpene production or biosynthetic pathway elucidation. The design developed here can be applied to the production of any GGPP-derived isoprenoid and is compatible with other yeast terpene production platforms.

Overexpression of erg20 gene encoding farnesyl pyrophosphate synthase has contrasting effects on activity of enzymes of the dolichyl and sterol branches of mevalonate pathway in Trichoderma reesei.

The mevalonate pathway is the most diverse metabolic route resulting in the biosynthesis of at least 30,000 isoprenoid compounds, many of which, such as sterols or dolichols, are indispensable for living cells. In the filamentous fungus Trichoderma of major biotechnological interest isoprenoid metabolites are also involved in the biocontrol processes giving the mevalonate pathway an additional significance. On the other hand, little is known about genes coding for enzymes of the mevalonate pathway in Trichoderma. Here, we present cloning and functional analysis of the erg20 gene from Trichoderma reesei coding for farnesyl pyrophosphate (FPP) synthase (EC 2.5.1.10), an enzyme located at the branching point of the mevalonate pathway. Expression of the gene in a thermosensitive erg20-2 mutant of Saccharomyces cerevisiae impaired in the FPP synthase activity suppressed the thermosensitive phenotype. The same gene overexpressed in T. reesei significantly enhanced the FPP synthase activity and also stimulated the activity of cis-prenyltransferase, an enzyme of the dolichyl branch of the mevalonate pathway. Unexpectedly, the activity of squalene synthase from the other, sterol branch, was significantly decreased without, however, affecting ergosterol level.

GGPPS1 predicts the biological character of hepatocellular carcinoma in patients with cirrhosis.

BACKGROUND: Hepatocellular carcinoma (HCC) has been associated with diabetes and obesity, but a possible connection with the metabolic syndrome (MetS) and its potential interaction with hepatitis and cirrhosis are open to discussion. Our previous investigations have shown that GGPPS1 plays a critical role during hyperinsulinism. In this report, the expression and distribution of GGPPS1 in liver cancer, and its clinical significance were investigated. METHODS: 70 patients with hepatocellular carcinoma (HCC) were included in this study. Three different types of tissues from each HCC patient were assembled immediately after surgical resection: tumor-free tissue >5 cm far from tumor edge (TF), adjacent nonmalignant tissue within 2 cm (AT), and tissue from the tumor (TT). Normal liver tissues from 10 liver transplant donors served as healthy control (HC) while 10 patients with liver cirrhosis as cirrhosis control (CC). The expression and distribution of GGPPS1 were detected by immunohistochemistry, western blots, or real-time PCR. The relationship between the expression of GGPPS1 and clinic pathologic index were analyzed. RESULTS: We found that GGPPS1 was intensified mainly in the cytoplasm of liver tumor cells. Both the expression of GGPPS1 mRNA and protein were upregulated in TT comparing to AT or TF. Meanwhile, HCC patients with cirrhosis had relative higher expression of GGPPS1. In addition, many pathologic characters show close correlation with GGPPS1, such as tumor stage, vessel invasion, and early recurrence. CONCLUSION: GGPPS1 may play a critical role during the development of HCC from cirrhosis and is of clinical significance for predicting biological character of HCC.

Upregulation of endogenous farnesyl diphosphate synthase overcomes the inhibitory effect of bisphosphonate on protein prenylation in Hela cells.

Nitrogen-containing bisphosphonates (N-BPs) such as zoledronic acid (ZOL) are the gold standard treatment for diseases of excessive bone resorption. N-BPs inactivate osteoclasts via inhibition of farnesyl diphosphate synthase (FPPS), thereby preventing the prenylation of essential small GTPases. Not all patients respond to N-BP therapy to the same extent, and some patients, for example with tumour-associated bone disease or Paget's disease, appear to develop resistance to N-BPs. The extent to which upregulation of FPPS might contribute to these phenomena is not clear. Using quantitative PCR and western blot analysis we show that levels of FPPS mRNA and protein can be upregulated in HeLa cells by culturing in lipoprotein deficient serum (LDS) or by over-expression of SREBP-1a. Upregulated, endogenous FPPS was predominantly localised to the cytosol and did not co-localise with peroxisomal or mitochondrial markers. Upregulation of endogenous FPPS conferred resistance to the inhibitory effect of low concentrations of ZOL on the prenylation of the small GTPase Rap1a. These observations suggest that an increase in the expression of endogenous FPPS could confer at least partial resistance to the pharmacological effect of N-BP drugs such as ZOL in vivo.

Engineered heterologous FPP synthases-mediated Z,E-FPP synthesis in E. coli.

Production of Z-type farnesyl diphosphate (FPP) has not been reported in Escherichia coli. Here we present the fusion enzyme (ILRv) of E. coli E,E-FPP synthase (IspA) and Mycobacterium tuberculosis Z,E-FPP synthase (Rv1086), which can produce primarily Z,E-FPP rather than E,E-FPP, the predominant stereoisomer found in most organisms. Z,E-farnesol (FOH) was produced from E. coli harboring the bottom portion of the MVA pathway and the fusion FPP synthase (ILRv) at a titer of 115.6 mg/L in 2YT medium containing 1% (v/v) glycerol as a carbon source and 5 mM mevalonate. The Z,E-FOH production was improved by 15-fold, compared with 7.7 mg/L obtained from the co-overexpression of separate IspA and Rv1086. The Z,E-FPP was not metabolized in native metabolic pathways of E. coli. It would be of interest to produce Z,E-FPP derived sesquiterpenes from recombinant E. coli due to no loss of Z,E-FPP substrate in endogenous metabolism of the host strain.

Molecular cloning and characterization of three isoprenyl diphosphate synthase genes from alfalfa.

Isoprenoid is the precursor for the biosynthesis of saponins, abscisic acid, gibberellins, chlorophylls and many other products in plants. Saponins are an important group of bioactive plant natural products. The alfalfa (Medicago sativa L.) saponins are glycosides of different triterpene aglycones and possess many biological activities. We isolated three genes (MsFPPS, MsGPPS and MsGGPPS) encoding isoprenyl diphosphate synthases (IDS) from alfalfa via a homology-based PCR approach. The enzyme activity assay of purified recombined MsFPPS and MsGGPPS expressed in Escherichia coli indicated that they all had IDS activity. Expression analysis of the three genes in different alfalfa tissues using real time PCR displayed that they were expressed in all tissues although they had a different expression patterns. MsFPPS and MsGPS displayed a significant increase in transcript level in response to methyl jasmonate, but the transcript level of MsGGPPS decreased obviously. To elucidate the functions of the three IDSs, their overexpression driven by a constitutive cauliflower mosaic virus-35S promoter in tobacco plants was applied and analyzed. The T(0) transgenic plants of MsFPPS showed high levels of squalene content when compared with control. However, no differences were detected in T(0) transgenic plants of MsGPPS and MsGGPPS. In addition, the overexpression of MsFPPS induced senescence response in transgenic plant leaves. This result may indicate that MsFPPS performs a role not only in phytosterol and triterpene biosynthesis, but also in growth regulation.

Sterol-regulatory-element-binding protein 2 and nuclear factor Y control human farnesyl diphosphate synthase expression and affect cell proliferation in hepatoblastoma cells.

FDPS (farnesyl diphosphate synthase) catalyses the formation of farnesyl diphosphate, a key intermediate in the synthesis of cholesterol and isoprenylated cellular metabolites. FDPS is also the molecular target of nitrogen-containing bisphosphonates, which are used as bone-antiresorptive drugs in various disorders. In the present study, we characterized the sterol-response element and NF-Y (nuclear factor Y)-binding site in the human FDPS promoter. Using a luciferase assay, electrophoretic mobility-shift assay and chromatin immunoprecipitation assay, we demonstrated that these elements are responsible for the transcription of the FDPS gene, and that its transcriptional activation is mediated by SREBP-2 (sterol-regulatory-element-binding protein 2) and NF-Y. We also investigated whether sterol-mediated FDPS expression is involved in the cell proliferation induced by zoledronic acid, an FDPS inhibitor. We show that the SREBP-2- and NF-Y-mediated regulation of FDPS gene transcription modulates cell proliferation. These results suggest that SREBP-2 and NF-Y are required to trigger cell proliferation through the induction of FDPS expression and that the pharmacological action of zoledronic acid is involved in this pathway.

Experimental study on the suppression of human nuclear receptor hLRH-1 via a vector-based RNA interference.

To explore the inhibitions of human nuclear receptor hLRH-1 via RNA interference, siRNAs expressing vectors pShLRH-1.1 and pShLRH-1.2, and targeting hLRH-1 were designed and constructed. The recombinants were introduced into hepatocellular carcinoma cells, BEL-7402, mediated by lipofectamin. RT-PCR was carried out to examine the inhibition ratio of hLRH-1 expression. The same method was also applied to analyze the expression of farnesyl pyrophosphate synthetase (FPPS) gene. Our results demonstrated that after transient transfection, both pShLRH-1.1 and pShLRH-1.2 could trigger the efficient inhibition of hLRH-1 in cultured cells, BEL-7402. The inhibition ratios were up to 80%. By comparing with non-transfection and vector-transfection control, the expression of FPPS in cells with inhibition of hLRH-1 was up-regulated significantly. Thus, the inhibition of expression of hLRH-1 in cultured cells was achieved via RNA interference in this study. Our results also suggested that hLRH-1 acts as a negative regulator in FPPS expression.