Intracellular Product Accumulation Research Articles

ASTAXANTHIN SYNTHESIS IN SYNECHOCOCCUS PC7002: OPTIMIZATION AND PRODUCTSECRETIONTO CULTURE MEDIUM

Synechococcus sp. PCC7002 can produce theastaxanthin precursor β-carotene but not astaxanthin.Using a synthetic ketolase-and a synthetic hydroxylase-gene we modifiedSynechococcuscarotene metabolism to produce astaxanthin. The total cellular content of astaxanthin was further increased by over-expressing the intrinsic phytoene synthase. Besides a beneficial step in astaxanthin production our results indicate for some modifications in the suggestedprocess of carotenoid metabolism. In addition, we were expressing an E. coli ABC-transporter to export astaxanthinand decrease intracellular product accumulation. By this, the synthesis was increased further and significant amounts of astaxanthin can be recovered from culture medium without cell destruction.

Development of morphology engineering for production of bio-based chemicals

Synthetic biology and metabolic engineering have been widely used to construct microbial cell factories for efficient production of bio-based chemicals, which mainly focus on the modification and regulation of metabolic pathways. The characteristics of microorganisms themselves, e.g. morphology, have rarely been taken into consideration in the biotechnological production processes. Morphology engineering aims to control cell shapes and cell division patterns by manipulating the genes related to cell morphology, providing a new strategy for developing efficient microbial cell factories. This review summarized the proteins related to cell morphology, followed by illustrating a few examples of using morphology engineering strategies for improving production of bio-based chemicals. This includes increasing intracellular product accumulation by regulating cell size, enhancing extracellular secretion of target products by improving cell permeability, reducing production cost by achieving high cell density, and improving product performance by controlling the degree of product hydrolysis. Finally, challenges and perspectives for the development of morphology engineering were discussed.

Production and secretion dynamics of prokaryotic Penicillin G acylase in Pichia pastoris

To take full advantage of recombinant Pichia pastoris (Komagataella phaffii) as a production system for heterologous proteins, the complex protein secretory process should be understood and optimised by circumventing bottlenecks. Typically, little or no attention has been paid to the fate of newly synthesised protein inside the cell, or its passage through the secretory pathway, and only the secreted product is measured. However, the system’s productivity (i.e. specific production rate qp), includes productivity of secreted (qp,extra) plus intracellularly accumulated (qp,intra) protein. In bioreactor cultivations with P. pastoris producing penicillin G acylase, we studied the dynamics of product formation, i.e. both the specific product secretion (qp,extra) and product retention (qp,intra) as functions of time, as well as the kinetics, i.e. productivity in relation to specific growth rate (μ). Within the time course, we distinguished (I) an initial phase with constant productivities, where the majority of product accumulated inside the cells, and qp,extra, which depended on μ in a bell-shaped manner; (II) a transition phase, in which intracellular product accumulation reached a maximum and productivities (intracellular, extracellular, overall) were changing; (III) a new phase with constant productivities, where secretion prevailed over intracellular accumulation, qp,extra was linearly related to μ and was up to three times higher than in initial phase (I), while qp,intra decreased 4–6-fold. We show that stress caused by heterologous protein production induces cellular imbalance leading to a secretory bottleneck that ultimately reaches equilibrium. This understanding may help to develop cultivation strategies for improving protein secretion from P. pastoris.Key Points• A novel concept for industrial bioprocess development.• A Relationship between biomass growth and product formation in P. pastoris.• A Three (3) phases of protein production/secretion controlled by the AOX1-promoter.• A Proof of concept in production of industrially relevant penicillin G acylase.

Disruption of vacuolar protein sorting components of the HOPS complex leads to enhanced secretion of recombinant proteins in Pichia pastoris

BackgroundThe yeast Pichia pastoris is a widely used host for the secretion of heterologous proteins. Despite being an efficient producer, we observed previously that certain recombinant proteins were mistargeted to the vacuole on their route to secretion. Simultaneous disruption of one vacuolar sorting pathway together with vacuolar proteases prevented this mis-sorting and resulted in higher levels of secreted heterologous protein. Inspired by the positive results, we now set out to investigate the influence of further parts of the vacuolar pathway, namely the Cvt-pathway and the homotypic fusion and protein sorting (HOPS) complex.ResultsStrains impaired in the Cvt pathway (∆atg11, ∆atg8) had no effect on secretion of the model protein carboxylesterase (CES), but resulted in lower secretion levels of the antibody fragment HyHEL-Fab. Disruption of genes involved in the HOPS complex led to vacuole-like compartments of the B category of vps mutants, which are characteristic for the deleted genes YPT7, VPS41 and VAM6. In particular ∆ypt7 and ∆vam6 strains showed an improvement in secreting the model proteins HyHEL-Fab and CES. Additional disruption of the vacuolar protease Pep4 and the potential protease Vps70 led to even further enhanced secretion in ∆ypt7 and ∆vam6 strains. Nevertheless, intracellular product accumulation was still observed. Therefore, the secretory route was strengthened by overexpression of early or late secretory genes in the vacuolar sorting mutants. Thereby, overexpression of Sbh1, a subunit of the ER translocation pore, significantly increased HyHEL-Fab secretion, leading up to fourfold higher extracellular Fab levels in the ∆ypt7 strain. The beneficial impact on protein secretion and the suitability of these strains for industrial applicability was confirmed in fed-batch cultivations.ConclusionsDisruption of genes involved in the HOPS complex, especially YPT7, has a great influence on the secretion of the two different model proteins HyHEL-Fab and CES. Therefore, disruption of HOPS genes shows a high potential to increase secretion of other recombinant proteins as well. Secretion of HyHEL-Fab was further enhanced when overexpressing secretion enhancing factors. As the positive effect was also present in fed-batch cultivations, these modifications likely have promising industrial relevance.

Optimization of process parameters by response surface methodology and kinetic modeling for batch production of canthaxanthin by Dietzia maris NIT-D (accession number: HM151403)

Dietzia maris NIT-D, a canthaxanthin producer, was isolated during routine screening of pigment-producing bacteria. Response surface methodology was applied for statistical designing of process parameters for biomass and canthaxanthin production. The effects of four process parameters (considered as independent variables), namely temperature (10-30°C), pH (4.75-5.75), shaker speed (75-135rpm) and percentage inoculum (0.5-2.5%) on the biomass and canthaxanthin yield (considered as dependent variables) were studied. As much as 122mgL(-1) of canthaxanthin was obtained when Dietzia maris NIT-D was incubated for 120h at 25°C and 120rpm, initial pH and percentage inoculum being 5.5 and 2% respectively. The pigment yield is the highest reported till date, with Dietzia maris as the test organism. The maximum biomass yield was 7.39gL(-1) under optimized process parameters. The predicted values were also verified by validation experiments in 5-day fermentation. Different mathematical models were used to describe growth and production, considering the effect of glucose in batch mode. The kinetic constants were calculated by fitting the experimental data to the models. Cell growth was inhibited beyond a glucose concentration of 15gL(-1). Andrews' model gave the best fit with a R (2) value of 0.9993. During the exponential growth phase, the specific growth rate was found to remain fairly constant with respect to time. There was no inhibitory effect due to intracellular product accumulation for all concentrations of glucose. This observation is the first of its kind, as previous studies have reported that increasing accumulation of intracellular carotenoid exerts greater degree of inhibition on growth.

Fedbatch design for periplasmic product retention in Escherichia coli

The feed profile of glucose during fedbatch cultivation could be used to influence the retention of the periplasmic product ZZ-cutinase. An increased feed rate led to a higher production rate but also to an increased specific leakage, which reduced the periplasmic retention. Three growth rates: 0.3, 0.2 and 0.1 h −1 where studied and resulted in 20, 9 and 6%, respectively, of the total ZZ-cutinase accumulating in the medium. It was also shown that leakage during fedbatch production of a Fab fragment was also influenced by the feed rate in a similar manner to ZZ-cutinase. If intracellular product accumulation is desired the advantage of a high productivity, resulting from a high substrate feed rate, is diminished because of a reduced product retention. Biochemical analysis revealed that the growth rate, resulting from a glucose limited feed, influenced the outer membrane protein compositions with respect to OmpF and LamB, whilst OmpA was largely unaffected. As the feed rate increased the amount of total outer membrane protein decreased. When ZZ-cutinase was produced there were further reductions in outer membrane protein accumulation, by 82, 100 and 22% for OmpF, LamB and OmpA, respectively, and the total reduction was almost 60% with a high product formation rate. We suggest that the reduced titre of the outer membrane proteins, OmpF and LamB, may have contributed to a reduced ability for the cell to retain recombinant protein secreted to the periplasm.

Permeability issues in whole-cell bioprocesses and cellular membrane engineering

Nutrient uptake and waste excretion are among the many important functions of the cellular membrane. While permitting nutrients into the cell, the cellular membrane system evolves to guide against noxious agents present in the environment from entering the intracellular milieu. The semipermeable nature of the membrane is at odds with biomolecular engineers in their endeavor of using microbes as cell factory. The cellular membrane often retards the entry of substrate into the cellular systems and prevents the product from being released from the cellular system for an easy recovery. Consequently, productivities of whole-cell bioprocesses such as biocatalysis, fermentation, and bioremediations are severely compromised. For example, the rate of whole-cell biocatalysis is usually 1-2 orders of magnitude slower than that of the isolated enzymes. When product export cannot keep pace with the production rate, intracellular product accumulation quickly leads to a halt of production due to product inhibition. While permeabilization via chemical or physical treatment of cell membrane is effective in small-scale process, large-scale implementation is problematic. Molecular engineering approach recently emerged as a much better alternative. Armed with increasingly sophisticated tools, biomolecular engineers are following nature's ingenuity to derive satisfactory solutions to the permeability problem. This review highlights these exciting molecular engineering achievements.

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.

Model-based fed-batch cultivation of R. eutropha for enhanced biopolymer production

Batch kinetics of the PHB synthesis was established in bioreactor under controlled environmental conditions of pH and dissolved oxygen. The data of the batch kinetics was used to develop and identify a mathematical model. The nutrient feeding strategies in the fed-batch cultivation were developed aiming at maintenance of limiting nutrient for either growth enhancement or increased intracellular product accumulation by offline computer simulation of mathematical model. The selected fed-batch fermentation strategies, using the predicted feeding profiles, were experimentally implemented to improve the growth/product formation/productivity and establish the validity of the developed model. The model predictions were very close to the observed experimental data points.

Analysis of growth rate effects on productivity of recombinant Escherichia coli populations using molecular mechanism models.

The influence of growth rate on Escherichia coli plasmid content and expression of a cloned-gene product has been described by a mathematical model based upon the molecular mechanism of lambdadv plasmid replication and known relationships between growth rate and transcription and translation activities of the host cell. The model simulates correctly decreases in plasmid content with increasing growth rate as observed experimentally for pBR322, NR1, R1, and Col E1 plasmids. A maximum with respect to growth rate in intracellular product accumulation is indicated by the model, as is a transient overshoot in product concentration following a shift from smaller to larger growth rate. Available data, although very limited, show the same trends. These results, obtained without parameter or kinetic form adjustments or manipulation, clearly illustrate the advantages of kinetic descriptions of recombinant systems based upon the pertinent molecular mechanisms.

Analysis of growth rate effects on productivity of recombinant Escherichia coli populations using molecular mechanism models. Reprinted from Biotechnology and Bioengineering, Vol. 26, Issue 1, Pages 66-73 (1984).

The influence of growth rate on Escherichia coli plasmid content and expression of a cloned-gene product has been described by a mathematical model based upon the molecular mechanism of lambdadv plasmid replication and known relationships between growth rate and transcription and translation activities of the host cell. The model simulates correctly decreases in plasmid content with increasing growth rate as observed experimentally for pBR322, NR1, R1, and Col E1 plasmids. A maximum with respect to growth rate in intracellular product accumulation is indicated by the model, as is a transient overshoot in product concentration following a shift from smaller to larger growth rate. Available data, although very limited, show the same trends. These results, obtained without parameter or kinetic form adjustments or manipulation, clearly illustrate the advantages of kinetic descriptions of recombinant systems based upon the pertinent molecular mechanisms.

Microbial Conversion of Methane into poly-β-hydroxybutyrate (PHB): Growth and intracellular product accumulation in a type II methanotroph

The biochemical pathways of methane to poly-β-hydroxybutyrate (PHB) in type II methanotrophs have been analyzed and used to propose stoichiometric equations for cell biomass and PHB. Conditions necessary for PHB accumulation in a batch culture of Methylocystis parvus OBBP were studied. In nitrogen limited cultures PHB started accumulating in the declining growth phase and maximum rate of PHB formation occurred during the late growth and early stationary phases. Inoculum age had an effect on maximum level of intracellular PHB, which increased with inoculum age up to ca. 70% of the cell dry weight using a 70 h inoculum. It has been shown that oxygen and methane requirements are high and pose a great stress on the mass transfer in the system; this limits the obtainment of high cell concentrations. When conditions for increased mass transfer of both CH 4 and O 2 were used, 5 g/ l of cells could be produced. Finally an enrichment strategy was investigated so that cells would start accumulating PHB earlier during the growth phase. Alternatives to increase cell mass concentrations are discussed.

Analysis of growth rate effects on productivity of recombinant Escherichia coli populations using molecular mechanism models

The influence of growth rate on Escherichia coli plasmid content and expression of a cloned-gene product has been described by a mathematical model based upon the molecular mechanism of lambdadv plasmid replication and known relationships between growth rate and transcription and translation activities of the host cell. The model simulates correctly decreases in plasmid content with increasing growth rate as observed experimentally for pBR322, NR1, R1, and Col E1 plasmids. A maximum with respect to growth rate in intracellular product accumulation is indicated by the model, as is a transient overshoot in product concentration following a shift from smaller to larger growth rate. Available data, although very limited, show the same trends. These results, obtained without parameter or kinetic form adjustments or manipulation, clearly illustrate the advantages of kinetic descriptions of recombinant systems based upon the pertinent molecular mechanisms.

Intracellular product accumulation in biotin-restricted yeast

The cells of biotin-restricted yeasts contain 5-aminoimidazole (or its riboside), inosine, and deamido-DPN. In addition to these substances, hypoxanthine and nicotinic acid have been reported in the medium, but are not detectable within the cells. If the biotin-restricted yeasts are supplied with aspartate or biotin, the amount of these materials is markedly decreased.