Menghao Cai

Mit1 Transcription Factor Mediates Methanol Signaling and Regulates the Alcohol Oxidase 1 (AOX1) Promoter in Pichia pastoris

The alcohol oxidase 1 (AOX1) promoter (PAOX1) of Pichia pastoris is the most powerful and commonly used promoter for driving protein expression. However, mechanisms regulating its transcriptional activity are unclear. Here, we identified a Zn(II)2Cys6-type methanol-induced transcription factor 1 (Mit1) and elucidated its roles in regulating PAOX1 activity in response to glycerol and methanol. Mit1 regulated the expression of many genes involved in methanol utilization pathway, including AOX1, but did not participate in peroxisome proliferation and transportation of peroxisomal proteins during methanol metabolism. Structural analysis of Mit1 by performing domain deletions confirmed its specific and critical role in the strict repression of PAOX1 in glycerol medium. Importantly, Mit1, Mxr1, and Prm1, which positively regulated PAOX1 in response to methanol, were bound to PAOX1 at different sites and did not interact with each other. However, these factors cooperatively activated PAOX1 through a cascade. Mxr1 mainly functioned during carbon derepression, whereas Mit1 and Prm1 functioned during methanol induction, with Prm1 transmitting methanol signal to Mit1 by binding to the MIT1 promoter (PMIT1), thus increasingly expressing Mit1 and subsequently activating PAOX1.

Identified biosynthetic pathway of aspergiolide A and a novel strategy to increase its production in a marine-derived fungus Aspergillus glaucus by feeding of biosynthetic precursors and inhibitors simultaneously

Aspergiolide A is a novel anti-tumor anthraquinone derivant produced by marine-derived fungus Aspergillus glaucus. To identify its biosynthetic pathway and further improve the production, the effects of biosynthetic pathway specific inhibitors and precursors were investigated. Cerulenin and iodoacetamide, the specific inhibitors of polyketide pathway, could completely inhibit the aspergiolide A accumulation. Putative precursors of polyketide pathway could increase aspergiolide A production greatly, such as 6 mM acetate increased production by 135%. Simvastatin and citrate, the inhibitors of mevalonate pathway, stimulated the production by 63% and 179%, respectively. Considering that acetyl-CoA is the common starter unit in both polyketide and mevalonate pathway, a novel strategy was designed to stimulate the aspergiolide A accumulation. Combinations of 12 mM acetate with 0.3 mM simvastatin could increase the production by 151%, while the supplementation with 12 mM acetate and 12 mM citrate brought a 262% increase of aspergiolide A production. The strategy might be very useful to enhance the production of other secondary metabolites derived from polyketide pathway.

Methanol-Independent Protein Expression by AOX1 Promoter with trans -Acting Elements Engineering and Glucose-Glycerol-Shift Induction in Pichia pastoris

The alcohol oxidase 1 promoter (PAOX1) of Pichia pastoris is commonly used for high level expression of recombinant proteins. While the safety risk of methanol and tough process control for methanol induction usually cause problems especially in large-scale fermentation. By testing the functions of trans-acting elements of PAOX1 and combinatorially engineering of them, we successfully constructed a methanol-free PAOX1 start-up strain, in which, three transcription repressors were identified and deleted and, one transcription activator were overexpressed. The strain expressed 77% GFP levels in glycerol compared to the wide-type in methanol. Then, insulin precursor (IP) was expressed, taking which as a model, we developed a novel glucose-glycerol-shift induced PAOX1 start-up for this methanol-free strain. A batch phase with glucose of 40 g/L followed by controlling residual glucose not lower than 20 g/L was compatible for supporting cell growth and suppressing PAOX1. Then, glycerol induction was started after glucose used up. Accordingly, an optimal bioprocess was further determined, generating a high IP production of 2.46 g/L in a 5-L bioreactor with dramatical decrease of oxygen consumption and heat evolution comparing with the wild-type in methanol. This mutant and bioprocess represent a safe and efficient alternative to the traditional glycerol-repressed/methanol-induced PAOX1 system.

Enhancing aspergiolide A production from a shear-sensitive and easy-foaming marine-derived filamentous fungus Aspergillus glaucus by oxygen carrier addition and impeller combination in a bioreactor.

Production enhancement of a novel antitumor compound aspergiolide A from shear-sensitive and easy-foaming marine-derived fungus Aspergillus glaucus HB1-19 in a 5-l stirred bioreactor was investigated. Two types of impellers, i.e., six-flat-blade disc turbine impeller (DT) and three-sector-blade pitched blade turbine impeller (PB) were used in this work. In cultures with fermentation medium, the combination of upper PB and lower DT led to the maximum dry biomass (13.8 g/l) and aspergiolide A production (19.3 mg/l). However, two PBs brought the highest aspergiolide A yield coefficient (1.9 mg/g dry biomass) despite it produced the lowest dry biomass (5.3 g/l). By contrast, two DTs and the upper DT and lower PB showed insignificant results. Feeding 0.35% (v/v) n-dodecane in cultures with upper PB and lower DT further improved aspergiolide A production by 31.0%, i.e., 25.3 mg/l, which is also 322% higher than that in the ordinary cultures with two DTs.

Efficient strategy for enhancing aspergiolide A production by citrate feedings and its effects on sexual development and growth of marine-derived fungus Aspergillus glaucus.

Aspergiolide A production enhancement by citrate and its effects on growth and sexual development of marine-derived fungus Aspergillus glaucus HB1-19 were investigated. In agar plate culture, 15 mM citric acid decreased colony radial growth and aspergiolide A production by 31.5% and 23.0%, respectively. It also improved sexual cleistothecium formation by 360% but depressed asexual conidiospore generation by 84.8%. In submerged culture, adding 40 mM citric acid finally promoted aspergiolide A production by 80.0%, which accompanied with 16.7% increase of biomass and 10.0% enhancement of sugar utilization. Differently, sodium citrate made no obvious or even opposite effects. Citrate and low pH could significantly improve pyruvate accumulation but inhibit succinate and fumarate production. Moreover, low pH was favorable to citrate utilization. Organic acids changes were closely related to aspergiolide A biosynthesis. Comparing to pH controls, effects of citric acid comprised pH decrease solicitation and citrate utilization enhancement.

Characterization of a thermostable raw-starch hydrolyzing α-amylase from deep-sea thermophile Geobacillus sp.

A deep-sea thermophile, Geobacillus sp. 4j, was identified to grow on starch and produce thermostable amylase. N-terminally truncated form of Geobacillus sp. 4j α-amylase (Gs4j-amyA) was fused at its N-terminal end with the signal peptide of outer membrane protein A (OmpA) of Escherichia coli. The enzyme was over-expressed in E. coli BL21 with a maximum extracellular production of 130U/ml in shake flask. The yield of the transformant increased 22-fold as compared with that of the wild strain. The recombinant enzyme purified to apparent homogeneity by metal-affinity chromatography, exhibited a molecular mass of 62kDa. It displayed the maximal activity at 60-65°C and pH 5.5. Its half-life (t1/2) at 80°C was 4.25h with a temperature deactivation energy of 166.3kJ/mol. Compared to three commonly used commercial α-amylases, the Gs4j-amyA exhibited similar thermostable performance to BLA but better than BAA and BSA. It also showed a universally efficient raw starch hydrolysis performance superior to commercial α-amylases at an acidic pH approaching nature of starch slurry. As a new acidic-resistant thermostable α-amylase, it has the potential to bypass the industrial gelatinization step in raw starch hydrolysis.

Biosynthetic origin of the carbon skeleton of a novel anti-tumor compound, haloroquinone, from a marine-derived fungus, Halorosellinia sp.

Purpose of work:The biosynthetic pathway of a new antitumor compound, haloroquinone, is elucidated to facilitate metabolic regulation for product accumulation and modification to produce new bioactive structural analogues of the compound.The biosynthetic origin of a novel promising protein kinase B inhibitor and anti-tumor compound, haloroquinone, from a marine-derived fungus, Halorosellinia sp. was clarified. The origin of carbon skeleton of haloroquinone was elucidated by feeding experiments with [2-13C]malonate and [1,2,3-13C3]malonate followed by 13C-NMR analysis of the isolated compounds: 15 carbon atoms were derived from malonate, of which eight were from the methylene group and seven from the carboxyl group. The remaining one is probably obtained by O-methylation. Haloroquinone is thus synthesized via a polyketide pathway using malonyl-CoA as both the starter unit and the extender unit.

A light–dark shift strategy derived from light-responded metabolic behaviors for polyketides production in marine fungus Halorosellinia sp.

Light, as an important environmental signal, generally brings about a broad regulation in fungal metabolism. In this work, we aim to explore the light-responded metabolic rules so as to further develop a feasible and effective light regulation strategy for production of anticancer polyketide 1403C by marine fungus Halorosellinia sp.. Light derived production enhancement of polyketides was first found in shake flask. To further understand this well working black box, light-responded cell growth, polyketides biosynthesis, metabolic behaviors (enzymes activities and organic acids levels) and mycelia morphology were then investigated in 5-L bioreactor. By comparing cultures under constant irradiation and dark conditions, the entire bioprocess was divided into two phases. During 0-60h, light presumably stimulated relevant metabolism to generate sufficient energy, NADPH and carbon skeleton, particularly malonyl-CoA, which was favorable for mycelia growth and polyketides accumulation. After 60h, light did harm to biomass and polyketides production. Consequently, a light-dark shift strategy was proposed and verified in 5-L bioreactor. It led to a maximal 1403C production of 1.67g/L, which was 24% and 74% higher than those obtained under constant irradiation and dark conditions, respectively.

Significant stimulation of o-phthalic acid in biosynthesis of aspergiolide A by a marine fungus Aspergillus glaucus.

The effect of o-phthalic acid (o-PA) on the production of the anti-tumor polyketide compound aspergiolide A by the marine fungus Aspergillus glaucus was investigated. o-PA at 12mM increased the aspergiolide A production by 77%. A combination of 12mM acetate with 12mM o-PA increased the production by 126%, i.e., from 27.1mg/l to 60.9mg/l, which was 27% and 75% higher than adding either 12mM o-PA or 12mM acetate, respectively. It was also found that A. glaucus could use o-PA as the sole carbon source on Minimal Medium. Ninety percent of o-PA was degraded by the fungus strain, especially when 12mM acetate was supplemented simultaneously. In culture medium supplemented with o-PA addition, sugar consumption decreased with increasing o-PA concentration, at the same time, the oxalacetate and pyruvate levels increased and the concentration of fumarate decreased. The results indicated that o-PA could stimulate biosynthesis of aspergiolide A effectively by regulating the metabolic pathway.

Engineered monoculture and co-culture of methylotrophic yeast for de novo production of monacolin J and lovastatin from methanol

As a promising one-carbon renewable substrate for industrial biotechnology, methanol has attracted much attention. However, engineering of microorganisms for industrial production of pharmaceuticals using a methanol substrate is still in infancy. In this study, the methylotrophic yeast Pichia pastoris was used to produce anti-hypercholesterolemia pharmaceuticals, lovastatin and its precursor monacolin J, from methanol. The biosynthetic pathways for monacolin J and lovastatin were first assembled and optimized in single strains using single copies of the relevant biosynthetic genes, and yields of 60.0mg/L monacolin J and 14.4mg/L lovastatin were obtained using methanol following pH controlled monoculture. To overcome limitations imposed by accumulation of intermediates and metabolic stress in monoculture, approaches using pathway splitting and co-culture were developed. Two pathway splitting strategies for monacolin J, and four for lovastatin were tested at different metabolic nodes. Biosynthesis of monacolin J and lovastatin was improved by 55% and 71%, respectively, when the upstream and downstream modules were separately accommodated in two different fluorescent strains, split at the metabolic node of dihydromonacolin L. However, pathway distribution at monacolin J blocked lovastatin biosynthesis in all designs, mainly due to its limited ability of crossing cellular membranes. Bioreactor fermentations were tested for the optimal co-culture strategies, and yields of 593.9mg/L monacolin J and 250.8mg/L lovastatin were achieved. This study provides an alternative method for production of monacolin J and lovastatin and reveals the potential of a methylotrophic yeast to produce complicated pharmaceuticals from methanol.