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This study investigated cell physiological and metabolic responses of Lactobacillus paracasei to osmotic stresses. Both cellular fatty acid composition and metabolite profiling were responded by increasing unsaturated and epoxy-fatty acid proportions, as well as accumulating some specific intracellular metabolites. Simultaneously, metabolite profiling was adopted to rationally and systematically discover potential osmoprotectants. Consequently, exogenous addition of proline or aspartate was validated to be a feasible and efficacious approach to improve cell growth under hyperosmotic stress in shake flasks. Particularly, with 5-L cultivation system, L-lactic acid concentration increased from 108 to 150 g/L during the following 16-h fermentation in 2 g/L proline addition group, while it only increased from 110 to 140 g/L in no proline addition group. Moreover, glucose consumption rate with proline addition reached 3.49 g/L/h during this phase, 35.8 % higher than that with no proline addition. However, extreme high osmotic pressure would significantly limit the osmoprotection of proline, and the osmolality threshold for L. paracasei was approximately 3600 mOsm/kg. It was suggested that proline principally played a role as a compatible solute accumulated in the cell for hyperosmotic preservation. The strategies of exploiting osmotic protectant with metabolite profiling and enhancing L-lactic acid production by osmoprotectant addition would be potential to provide a new insight for other microorganisms and organic acids production.

Penicillin is one of the best known pharmaceuticals and is also an important member of the β-lactam antibiotics. Over the years, ambitious yields, titers, productivities, and low costs in the production of the β-lactam antibiotics have been stepwise realized through successive rounds of strain improvement and process optimization. Penicillium chrysogenum was proven to be an ideal cell factory for the production of penicillin, and successful approaches were exploited to elevate the production titer. However, the industrial production of penicillin faces the serious challenge that environmental gradients, which are caused by insufficient mixing and mass transfer limitations, exert a considerably negative impact on the ultimate productivity and yield. Scale-down studies regarding diverse environmental gradients have been carried out on bacteria, yeasts, and filamentous fungi as well as animal cells. In accordance, a variety of scale-down devices combined with fast sampling and quenching protocols have been established to acquire the true snapshots of the perturbed cellular conditions. The perturbed metabolome information stemming from scale-down studies contributed to the comprehension of the production process and the identification of improvement approaches. However, little is known about the influence of the flow field and the mechanisms of intracellular metabolism. Consequently, it is still rather difficult to realize a fully rational scale-up. In the future, developing a computer framework to simulate the flow field of the large-scale fermenters is highly recommended. Furthermore, a metabolically structured kinetic model directly related to the production of penicillin will be further coupled to the fluid flow dynamics. A mathematical model including the information from both computational fluid dynamics and chemical reaction dynamics will then be established for the prediction of detailed information over the entire period of the fermentation process and thereby for the optimization of penicillin production, and subsequently also benefiting other fermentation products.

The recombinant Pichia pastoris harboring an improved methionine adenosyltransferase (MAT) shuffled gene was employed to biosynthesize S-adenosyl-L: -methionine (SAM). Two L: -methionine (L: -Met) addition strategies were used to supply the precursor: the batch addition strategy (L: -Met was added separately at three time points) and the continuous feeding strategies (L: -Met was fed continuously at the rate of 0.1, 0.2, and 0.5 g l(-1) h(-1), respectively). SAM accumulation, L: -Met conversion rate, and SAM productivity with the continuous feeding strategies were all improved over the batch addition strategy, which reached 8.46 +/- 0.31 g l(-1), 41.7 +/- 1.4%, and 0.18 +/- 0.01 g l(-1) h(-1) with the best continuous feeding strategy (0.2 g l(-1) h(-1)), respectively. The bottleneck for SAM production with the low L: -Met feeding rate (0.1 g L(-1) h(-1)) was the insufficient L: -Met supply. The analysis of the key enzyme activities indicated that the tricarboxylic acid cycle and glycolytic pathway were reduced with the increasing L: -Met feeding rate, which decreased the adenosine triphosphate (ATP) synthesis. The MAT activity also decreased as the L: -Met feeding rate rose. The reduced ATP synthesis and MAT activity were probably the reason for the low SAM accumulation when the L: -Met feeding rate reached 0.5 g l(-1) h(-1).

To develop an efficient way to produce S-adenosylmethionine (SAM), methionine adenosyltransferase gene (mat) from Streptomyces spectabilis and Vitreoscilla hemoglobin gene (vgb) were coexpressed intracellularly in Pichia pastoris, both under control of methanol-inducible promoter. Expression of mat in P. pastoris resulted in about 27 times higher specific activity of methionine adenosyltransferase (SMAT) and about 19 times higher SAM production relative to their respective control, suggesting that overexpression of mat could be used as an efficient method for constructing SAM-accumulating strain. Under induction concentration of 0.8 and 2.4% methanol, coexpression of vgb improved, though to different extent, cell growth, SAM production, and respiratory rate. However, the effects of VHb on SAM content (specific yield of SAM production) and SMAT seemed to be methanol concentration-dependent. When cells were induced with 0.8% methanol, no significant effects of VHb expression on SAM content and specific SMAT could be detected. When the cells were induced with 2.4% methanol, vgb expression increased SAM content significantly and depressed SMAT remarkably. We suggested that under our experimental scheme, the presence of VHb might improve ATP synthesis rate and thus improve cell growth and SAM production in the recombinant P. pastoris.

An S-adenosylmethionine synthetase (SAM-s) gene from Streptomyces spectabilis was integrated along with vector DNA into the chromosome of a Saccharopolyspora erythraea E2. Elevated production of SAM was observed in the recombinant strain Saccharopolyspora erythraea E1. The results from the bioassay showed that the titer of erythromycin was increased from 920 IU ml(-1) by E2 to approximately 2,000 IU ml(-1) by E1. High performance liquid chromatography (HPLC) analysis revealed that there was a 132% increase in erythromycin A compared with the original strain, while the erythromycin B, the main impurity component in erythromycin, was decreased by 30%. The sporulation process was inhibited, while the SAM-s gene was expressed. The addition of the exogenous SAM also inhibited sporulation and promoted an increase in erythromycin titers.

Lower induction temperature and polyoxyethylene sorbitan monolaurate (Tween-20) were successfully used to inhibit the aggregation of recombinant human consensus interferon-alpha mutant (cIFN) during Pichia pastoris fermentation. When the induction temperature was decreased from 30 to 20 degrees C, the cIFN secreted into the medium was in the form of monomers instead of aggregates. The maximum specific activity at 20 degrees C was 4.04 times as high as that at 30 degrees C. There was no obvious effect on the cell growth at 20 degrees C, but the total protein level was decreased. Similar inhibition effect on cIFN aggregation was observed when 0.2 g l(-1) Tween-20 was added during induction. Furthermore, there was a synergistic effect found between induction temperature and Tween-20 on the inhibition of cIFN aggregation. The maximum specific activity with Tween-20 at 20 degrees C was 19.9-fold higher than that without Tween-20 at 30 degrees C.

This minireview mainly aims at the study of S-adenosyl-L-methionine (SAM) production by microbial fermentation. A brief introduction of the biological role and application of SAM was presented. In general, SAM production can be improved by breeding of the producing strain through the conventional mutation or genetic engineering approach in the molecular or cellular scale, by optimization of culture conditions in the cellular scale or bioreactor engineering scale, or by multiscale approach. The productivity of SAM fermentation has been improved greatly through the efforts of many researchers using the methods previously mentioned. The SAM-producing strains used extensively are Pichia pastoris and Saccharomyces cerevisiae. The effect of SAM on antibiotic production was also exemplified. The skill and scheme beneficial to the improvement of SAM production involves the enhancement of SAM synthetase (methionine adenosyltransferase) activity and selection of engineered constitutive promoters with appropriate strength; seeking for and eliminating the rate-limiting factors in SAM synthesis, namely, knocking off the genes that transform SAM and L-methionine (L-Met) to cysteine; release the feedback inhibition of SAM to methylenetetrahydrofolate reductase; blocking the transsulfuration pathway by interfering the responsible enzymes; enhancing ATP level through pulsed feeding of glycerol; and optimizing the L-Met feeding strategy. Precise control of gene expression and quantitative assessment of physiological parameters in engineered P. pastoris were highlighted. Finally, a discussion of the prospect of SAM production was presented.

Increased copy number of foreign gene can result in the alteration of normal metabolism in Pichia pastoris. To better understand the effect of foreign gene dosage on the cellular physiology of P. pastoris cells, comparative transcriptional analysis was performed among three P. pastoris strains carrying 0, 6, and 18 copies of porcine insulin precursor (PIP) expression cassettes, respectively. mRNA levels of 13 selected genes involved in methanol metabolic pathway, central metabolic pathway, protein folding, and oxidative stress were determined by real-time PCR. Results showed that enhanced PIP copy number resulted in an increase in PIP mRNA and also in folding stress on the yeast cells’ endoplasmic reticulum. The metabolism of 6-copy P. pastoris strain was not significantly changed as compared to 0-copy strain (control). In contrast, physiology of 18-copy strain was remarkably affected, characterized by the upregulation of antioxidative genes and readjusted expression level of methanol metabolic pathway genes. These data suggested that high copy P. pastoris strain might be suffering from protein folding-related oxidative stress and insufficient supply of carbon and energy sources.

Previous study has shown that the degradation and aggregation of recombinant human consensus interferon-alpha mutant (cIFN) were serious when cIFN was secreted to bioreactor by Pichia pastoris. In this study, we showed that this phenomenon was concomitant well with the formation of the doublets of cIFN monomers that could be seen clearly on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The doublets were a mixture of two isomers formed by cIFN with different disulfide bonds and identified that the upper cIFN in doublets contains only one disulfide bond while the lower cIFN contains intact disulfide bonds by a novel method termed protein laddering map on SDS-PAGE. In addition, the instability of cIFN with different disulfide bond forms is also analyzed through a novel in vitro conversion assay based on incubation with different concentrations of beta-mercaptoethanol. The results showed that only a wound such as cleavage of only one disulfide bond could be fatal to cIFN stability. If the disulfide bonds in cIFN monomers were broken, three kinds of aggregates would be formed easily: covalent aggregates, non-covalent aggregates, and unknown dimers. Likewise, the unfolded species also displayed reduced stability to proteolysis. These results indicate that the incomplete formation of disulfide bond in cIFN secreted to fermentation broth triggers severe degradation and aggregation of cIFN, which result in sharp decrease of bioactivity of cIFN in bioreactor.

The methylotrophic yeast Pichia pastoris has been used for the expression of many proteins. However, limitations such as protein degradation and aggregation became obvious when secreting heterologous protein-recombinant human consensus interferon-alpha mutant. Here, we investigate the effect of induction temperature on the yield and stability of interferon mutant expressed by P. patoris with buffered complex medium. The best results in terms of interferon mutant bioactivity and specific bioactivity were obtained when the microorganism was induced at 15 degrees C, which were 2.91 x 10(8) +/- 0.3 x 10(8) and 2.26 x 10(8 )+/- 0.23 x 10(8) IU mg(-1), respectively. At the same time, the cells grew fast owing to high AOX1-specific activity, and interferon mutant expression level reached 1.23 g l(-1), which was almost 30 times higher than that in the flask. Also, the proteolytic degradation of interferon mutant was inhibited completely because of lower protease bioactivity probably due to a reduced cell death rate at lower temperatures as well as protection of yeast extract and peptone in complex medium. In addition, interferon mutant aggregation was repressed significantly by the addition of Tween-80, and a specific bioactivity of 7.35 x 10(8) +/- 0.56 x 10(8) IU mg(-1) was obtained. These results should be applicable to other low-stability recombinant proteins expressed in P. pastoris.