Rhizopus Oryzae Lipase Production Research Articles

Rhizopus oryzae Lipase, a Promising Industrial Enzyme: Biochemical Characteristics, Production and Biocatalytic Applications

Lipases are biocatalysts with a significant potential to enable a shift from current pollutant manufacturing processes to environmentally sustainable approaches. The main reason of this prospect is their catalytic versatility as they carry out several industrially relevant reactions as hydrolysis of fats in water/lipid interface and synthesis reactions in solvent-free or non-aqueous media such as transesterification, interesterification and esterification. Because of the outstanding traits of Rhizopus oryzae lipase (ROL), 1,3-specificity, high enantioselectivity and stability in organic media, its application in energy, food and pharmaceutical industrial sector has been widely studied. Significant advances have been made in the biochemical characterisation of ROL particularly in how its activity and stability are affected by the presence of its prosequence. In addition, native and heterologous production of ROL, the latter in cell factories like Escherichia coli, Saccharomyces cerevisiae and Komagataella phaffii (Pichia pastoris), have been thoroughly described. Therefore, in this review, we summarise the current knowledge about R. oryzae lipase (i) biochemical characteristics, (ii) production strategies and (iii) potential industrial applications.

Efficient Heterologous Production of Rhizopus oryzae Lipase via Optimization of Multiple Expression-Related Helper Proteins.

This study is dedicated to efficiently produce Rhizopus oryzae lipase (ROL) by optimizing the expression of multiple expression-related helper proteins in Pichia pastoris. A series of engineered strains harboring different copy numbers of the ROL gene and different copies of the chaperone Pdi gene were first constructed to examine the influence of Pdi gene copy number on ROL production. The results showed that multiple copies of Pdi gene did not significantly improve ROL expression. Then, the effect of the co-overexpression of 10 expression-related helper proteins on ROL secretion was investigated by screening 20 colonies of each transformants. The data from shaking-flask fermentation suggested that Ssa4, Bmh2, Sso2, Pdi, Bip, Hac1, and VHb had positive effects on ROL expression. Subsequently, Ssa4, Bmh2, and Sso2, which all participate in vesicular trafficking and strongly promote ROL expression, were combined to further improve ROL production level. ROL activity of the screened strain GS115/5ROL-Ssa4-Sso2-Bmh2 4# attained 5230 U/mL. Furthermore, when the helper proteins Pdi, Bip, Hac1, and VHb were individually co-expressed with ROL in the strain GS115/5ROL-Ssa4-Sso2-Bmh2 4#, lipase activity increased to 5650 U/mL in the strain GS115/5ROL-Ssa4-Sso2-Bmh2-VHb 9#. Additionally, the maximum ROL activity of 41,700 U/mL was achieved in a 3 L bioreactor for high-density fermentation via a sorbitol–methanol co-feeding strategy, reaching almost twofold the value of the initial strain GS115/pAOα-5ROL 11#. Thus, the strategies in this study significantly increased ROL expression level, which is of great potential for the large-scale production of ROL in P. pastoris.

The growth of Pichia pastoris Mut+ on methanol-glycerol mixtures fits to interactive dual-limited kinetics: model development and application to optimised fed-batch operation for heterologous protein production.

The methanol-glycerol co-feeding during the induction stage for heterologous protein production in Pichia pastoris has shown significant productive applications. Available model analysis applied to this dual-limited condition is scarce and normally does not consider the interaction effects between the substrates. In this work, a dual-limited growth model of P. pastoris considering an interactive kinetic effect was applied to an optimised fed-batch process production of heterologous Rhizopus oryzae lipase (ROL). In the proposed model, the growth kinetics on glycerol is fully expressed, whereas methanol kinetics is modulated by the co-metabolisation of glycerol, resulting in an enhancing effect of glycerol-specific growth rate. The modelling approach of fed-batch cultures also included the methanol volatilisation caused by the aeration that was found to be a not-negligible phenomenon. The model predicts the ability of P. pastoris to keep control of the methanol concentration in the broth during ROL-optimised production process in fed batch and fits satisfactorily the specific cell growth rate and ROL production. Implications of interaction effect are discussed applying the general procedure of modelling approach.

Bioprocess efficiency in Rhizopus oryzae lipase production by Pichia pastoris under the control of PAOX1 is oxygen tension dependent

Abstract Dissolved oxygen (DO) has been stated as a key parameter for Pichia pastoris (Mut+) cultures growing on methanol. DO set point (DOSP) – between 5 and 50% air saturation – with time-varying oxygen transfer rate (OTR) was investigated during the induction phase of fed batch cultures producing the heterologous Rhizopus oryzae lipase (ROL). DO around 25% was found to be the best operating condition in terms of ROL total productivity, with 4-fold increase in respect to DOSP of 5% (minimum value), and also corresponded with the highest specific rates of growth (μ), methanol consumption (qS), oxygen uptake (qO2), carbon dioxide production (qCO2) and ROL activity production (qP). However, the highest overall product yield (Y*P/X) was obtained around DOSP of 10%, with an increase of 45% compared with maximal DOSP studied (50%). Specific rates and Y*P/X were lower at the extremes of the DO range tested. The results validate that oxygen tension is essential to improve bioprocess efficiency in ROL production and enhances reliability of the system by keeping DO at optimal conditions. Productivity and yield can be optimized, thus reducing oxygen supply and energy requirements, even for different target proteins although presenting different DO optimal values.

Effect of dilution rate and methanol-glycerol mixed feeding on heterologous Rhizopus oryzae lipase production with Pichia pastoris Mut(+) phenotype in continuous culture.

The induction using substrate mixtures is an operational strategy for improving the productivity of heterologous protein production with Pichia pastoris. Glycerol as a cosubstrate allows for growth at a higher specific growth rate, but also has been reported to be repressor of the expression from the AOX1 promoter. Thus, further insights about the effects of glycerol are required for designing the induction stage with mixed substrates. The production of Rhizopus oryzae lipase (ROL) was used as a model system to investigate the application of methanol-glycerol feeding mixtures in fast metabolizing methanol phenotype. Cultures were performed in a simple chemostat system and the response surface methodology was used for the evaluation of both dilution rate and methanol-glycerol feeding composition as experimental factors. Our results indicate that productivity and yield of ROL are strongly affected by dilution rate, with no interaction effect between the involved factors. Productivity showed the highest value around 0.04-0.06 h(-1) , while ROL yield decreased along the whole dilution rate range evaluated (0.03-0.1 h(-1) ). Compared to production level achieved with methanol-only feeding, the highest specific productivity was similar in mixed feeding (0.9 UA g-biomass(-1) h(-1) ), but volumetric productivity was 70% higher. Kinetic analysis showed that these results are explained by the effects of dilution rate on specific methanol uptake rate, instead of a repressor effect caused by glycerol feeding. It is concluded that despite the effect of dilution rate on ROL yield, mixed feeding strategy is a proper process option to be applied to P. pastoris Mut(+) phenotype for heterologous protein production.

Searching the best operational strategies for Rhizopus oryzae lipase production in Pichia pastoris Mut+ phenotype: Methanol limited or methanol non-limited fed-batch cultures?

Two different operational strategies, methanol limited (MLFB) and methanol non-limited fed-batch (MNLFB) cultures, have been compared for the production of the recombinant Rhizopus oryzae lipase (ROL) expressed in Pichia pastoris Mut+ phenotype. Yields, productivities and specific production rate in all MLFB conditions were very low, obtaining the best results at the lower specific growth rates. Similar results were obtained for MNLFB strategy at methanol set-point concentrations up to 2gl−1. However, for methanol set-points higher than 2gl−1 a significant increase in the production was observed. Methanol set-point of 3gl−1 was the optimum to maximize product yield (YP/X), both volumetric and specific productivity and mean specific production rate (qP,mean), although in terms of ROL production high values were obtained in the range of 3–10gl−1. An inhibitory effect of methanol on cell growth as well as slowness on ROL production during early induction phase was observed at concentration of 10gl−1.

Engineering of bottlenecks in Rhizopus oryzae lipase production in Pichia pastoris using the nitrogen source-regulated FLD1 promoter

The yeast Pichia pastoris has been previously used for extracellular expression of a Rhizopus oryzae lipase (Rol). However, limitations in Rol folding and secretion through the cell wall became apparent when producing it in fed-batch cultivations. In this study, we have investigated the effect of combining two cell engineering strategies to alleviate putative bottlenecks in Rol secretion, namely the constitutive expression of the induced form of the Saccharomyces cerevisiae unfolded protein response transcriptional factor Hac1 and the deletion of the GAS1 gene encoding beta-1,3-glucanosyltransglycosylase, GPI-anchored to the outer leaflet of the plasma membrane, playing a key role in yeast cell wall assembly. The performance of these engineered Rol-producing strains has been compared in fed-batch cultivations set at a low specific growth rate of about 0.005 h-(1). It was found that Rol overexpression in a P. pastoris strain expressing constitutively the induced form of S. cerevisiae Hac1 and the deletion of GAS1 resulted in about a 3-fold and 4-fold increase in the overall process specific productivity, respectively, whereas the double mutant HAC1/deltagas1 strain yielded about a 7-fold increase. Overall, these results reflect the multiplicity of physiological bottlenecks at different levels/steps throughout the Rol synthesis, secretion and excretion processes in P. pastoris.

Transcriptional response of P. pastoris in fed-batch cultivations to Rhizopus oryzae lipase production reveals UPR induction

BackgroundThe analysis of transcriptional levels of the genes involved in protein synthesis and secretion is a key factor to understand the host organism's responses to recombinant protein production, as well as their interaction with the cultivation conditions. Novel techniques such as the sandwich hybridization allow monitoring quantitatively the dynamic changes of specific RNAs. In this study, the transcriptional levels of some genes related to the unfolded protein response (UPR) and central metabolism of Pichia pastoris were analysed during batch and fed-batch cultivations using an X-33-derived strain expressing a Rhizopus oryzae lipase under control of the formaldehyde dehydrogenase promoter (FLD1), namely the alcohol oxidase gene AOX1, the formaldehyde dehydrogenase FLD1, the protein disulfide isomerase PDI, the KAR2 gene coding for the BiP chaperone, the 26S rRNA and the R. oryzae lipase gene ROL.ResultsThe transcriptional levels of the selected set of genes were first analysed in P. pastoris cells growing in shake flask cultures containing different carbon and nitrogen sources combinations, glycerol + ammonium, methanol + methylamine and sorbitol + methylamine. The transcriptional levels of the AOX1 and FLD1 genes were coherent with the known regulatory mechanism of C1 substrates in P. pastoris, whereas ROL induction lead to the up-regulation of KAR2 and PDI transcriptional levels, thus suggesting that ROL overexpression triggers the UPR. This was further confirmed in fed-batch cultivations performed at different growth rates. Transcriptional levels of the analysed set of genes were generally higher at higher growth rates. Nevertheless, when ROL was overexpressed in a strain having the UPR constitutively activated, significantly lower relative induction levels of these marker genes were detected.ConclusionThe bead-based sandwich hybridization assay has shown its potential as a reliable instrument for quantification of specific mRNA species in P. pastoris cells grown in fed-batch cultures. As a proof-of-principle, the influence of the carbon and nitrogen sources, the specific growth rate, as well as the ROL overexpression on the transcriptional levels of a reduced set of bioprocess-relevant genes has been quantitatively studied, revealing that ROL overexpression and secretion seems to trigger the UPR in P. pastoris, resulting in a physiological bottleneck for the production process.

A simple model‐based control for Pichia pastoris allows a more efficient heterologous protein production bioprocess

A predictive control algorithm coupled with a PI feedback controller has been satisfactorily implemented in the heterologous Rhizopus oryzae lipase production by Pichia pastoris methanol utilization slow (Mut(s)) phenotype. This control algorithm has allowed the study of the effect of methanol concentration, ranging from 0.5 to 1.75 g/L, on heterologous protein production. The maximal lipolytic activity (490 UA/mL), specific yield (11,236 UA/g(biomass)), productivity (4,901 UA/L . h), and specific productivity (112 UA/g(biomass)h were reached for a methanol concentration of 1 g/L. These parameters are almost double than those obtained with a manual control at a similar methanol set-point. The study of the specific growth, consumption, and production rates showed different patterns for these rates depending on the methanol concentration set-point. Results obtained have shown the need of implementing a robust control scheme when reproducible quality and productivity are sought. It has been demonstrated that the model-based control proposed here is a very efficient, robust, and easy-to-implement strategy from an industrial application point of view.

State variables monitoring by in situ multi-wavelength fluorescence spectroscopy in heterologous protein production by Pichia pastoris

State variables throughout non-induced and induced cultivations of Pichia pastoris for the heterologous Rhizopus oryzae lipase (ROL) production were monitored with a multi-wavelength on-line fluorescence sensor. Based on this work, the use of in situ multi-wavelength fluorometry combined with chemometrics models (PLS-1 models) provided a quantitative prediction of biomass and substrates (glycerol and methanol) during non-induced and induced ROL production. The mean prediction errors for both variables were about 7% and 10%, respectively. ROL is also quite satisfactory estimated in the exponential growth phase with prediction errors similar to biomass and substrate variables. However, in the stationary phase, where proteolytic degradation of ROL is observed, the prediction error could get a value about 20%. This fact is due to the lower reproducibility of protein production from batch to batch.

Analysis of bottlenecks in Rhizopus oryzae lipase production in Pichia pastoris using the nitrogen source-regulated formaldehyde dehydrogenase promoter (PFLD1)

Background Methanol-free high cell density fed-batch cultivation strategies for the P. pastoris expression system have been recently developed by expressing a Rhizopus oryzae lipase (ROL) under the transcriptional control of the PFLD1 [1]. These cultivation strategies were based on the use of sorbitol and methylamine as carbon and nitrogen source, respectively, during the induction phase of the cultivation process. Fed-batch fermentations were performed at three different specific growth rates and showed that productivities were strongly correlated with this parameter (i.e. with the cell's physiological state). Moreover, intracellular active product accumulation and a decrease in the specific product secretion rate were observed along the induction phase of the fermentation process. These results suggested the presence of a bottleneck(s) throughout the synthesis and secretion process of the heterologous lipase.

Transcriptional analysis of protein production and induction of unfolded protein response in Pichia pastoris expressing a Rhizopus oryzae lipase under the FLD1 promoter

Background Methanol-free high cell density fed-batch cultivation strategies for the Pichia pastoris expression system have been recently developed by expressing a Rhizopus oryzae lipase (ROL) under the transcriptional control of FLD1 promoter (P FLD1)[1]. These cultivation strategies were based on the use of sorbitol and methylamine as carbon and nitrogen source, respectively, during the induction phase of the cultivation process. The specific growth rate proved to be an important parameter in the productivity of secreted ROL. Moreover, intracellular active product accumulation and a decrease in the specific product secretion rate were observed along the induction phase of the fermentation process. These results suggested the presence of a bottleneck(s) throughout the synthesis and secretion process of the heterologous lipase. Recently [2], flow cytometry analyses of intracellular ROL levels confirmed that a fraction of the product was retained within the cell. Further, this intracellular product accumulation was concomitant with an increase on the BiP protein, a chaperone of the HSP70 class that plays an important role in the unfolded protein response (UPR). Notably, the increase of BiP and ROL content in the cell was detected soon after the beginning of the induction phase. Interestingly, the intracellular BiP and ROL profiles were different depending on the specific growth rate of the cells. In this study, we report the application of a sandwich hybridization assay-based technique [3] for quantification of specific mRNAs levels during the extracellular production of ROL in P. pastoris under the transcriptional control of PFLD1. These studies have been carried out in fed-batch cultures at two different specific growth rates.