Glucose Fed-batch Fermentation Research Articles

Proteome reallocation enables the selective de novo biosynthesis of non-linear, branched-chain acetate esters

The one-carbon recursive ketoacid elongation pathway is responsible for making various branched-chain amino acids, aldehydes, alcohols, ketoacids, and acetate esters in living cells. Controlling selective microbial biosynthesis of these target molecules at high efficiency is challenging due to enzyme promiscuity, regulation, and metabolic burden. In this study, we present a systematic modular design approach to control proteome reallocation for selective microbial biosynthesis of branched-chain acetate esters. Through pathway modularization, we partitioned the branched-chain ester pathways into four submodules including ketoisovalerate submodule for converting pyruvate to ketoisovalerate, ketoacid elongation submodule for producing longer carbon-chain ketoacids, ketoacid decarboxylase submodule for converting ketoacids to alcohols, and alcohol acyltransferase submodule for producing branched-chain acetate esters by condensing alcohols and acetyl-CoA. By systematic manipulation of pathway gene replication and transcription, enzyme specificity of the first committed steps of these submodules, and downstream competing pathways, we demonstrated selective microbial production of isoamyl acetate over isobutyl acetate. We found that the optimized isoamyl acetate pathway globally redistributed the amino acid fractions in the proteomes and required up to 23–31% proteome reallocation at the expense of other cellular resources, such as those required to generate precursor metabolites and energy for growth and amino acid biosynthesis. From glucose fed-batch fermentation, the engineered strains produced isoamyl acetate up to a titer of 8.8 g/L (>0.25 g/L toxicity limit), a yield of 0.22 g/g (61% of maximal theoretical value), and 86% selectivity, achieving the highest titers, yields and selectivity of isoamyl acetate reported to date.

γ-PGA Fermentation by Bacillus subtilis PG-001 with Glucose Feedback Control pH-stat Strategy.

Poly-γ-glutamic acid (γ-PGA) is an important biopolymer with many applications due to its biodegradable and non-toxic characteristics. γ-PGA is produced industrially by fermentation of Bacillus species. The optimal pH range for producing γ-PGA by Bacillus subtilis PG-001 was firstly studied by glucose fed-batch fermentation with non-controlled pH. Result showed that both cell growth and γ-PGA synthesis were repressed when pH was lower than pH 6. Further investigation with γ-PGA fed-batch fermentation showed that pH 6.5 is more suitable for γ-PGA fermentation than pH 7. Under comparable consumption of glutamic acid and glucose, 11.8 g/L γ-PGA and 0.7 g/g yield were achieved by fermentation at pH 6.5, which was significantly higher than 10.5 g/L and 0.56 g/g yield of fermentation at pH 7. In addition, γ-PGA degradation during later phase of fermentation was repressed at pH 6.5 as 9238cP of final broth viscosity was achieved from fermentation at pH 6.5 while it was only 346 cP for fermentation at pH 7. Finally, a glucose feedback control pH-stat strategy was performed for reducing alkali consumption during γ-PGA fermentation, which further increased final γ-PGA concentration to 15.5 g/L with much higher viscosity (11458 cP); meanwhile the consumption of alkali decreased 57%. The fed-batch γ-PGA fermentation with glucose feedback control pH-stat strategy showed high feasibility for industrial scaling-up.

Rewiring central carbon metabolism for tyrosol and salidroside production in Saccharomyces cerevisiae.

Metabolic engineering of Saccharomyces cerevisiae for high-level production of aromatic chemicals has received increasing attention in recent years. Tyrosol production from glucose by S. cerevisiae is considered an environmentally sustainable and safe approach. However, the production of tyrosol and salidroside by engineered S. cerevisiae has been reported to be lower than 2 g/L to date. In this study, S. cerevisiae was engineered with a push-pull-restrain strategy to efficiently produce tyrosol and salidroside from glucose. The biosynthetic pathways of ethanol, phenylalanine, and tryptophan were restrained by disrupting PDC1, PHA2, and TRP3. Subsequently, tyrosol biosynthesis was enhanced with a metabolic pull strategy of introducing PcAAS and EcTyrAM53I/A354V . Moreover, a metabolic push strategy was implemented with the heterologous expression of phosphoketolase (Xfpk), and then erythrose 4-phosphate was synthesized simultaneously by two pathways, the Xfpk-based pathway and the pentose phosphate pathway, in S. cerevisiae. Furthermore, the heterologous expression of Xfpk alone in S. cerevisiae efficiently improved tyrosol production compared with the coexpression of Xfpk and phosphotransacetylase. Finally, the tyrosol yield increased by approximately 135-folds, compared with that of parent strain. The total amount of tyrosol and salidroside with glucose fed-batch fermentation was over 10 g/L and reached levels suitable for large-scale production.

Engineering Escherichia coli for methanol-dependent growth on glucose for metabolite production

Synthetic methylotrophy aims to engineer methane and methanol utilization pathways in platform hosts like Escherichia coli for industrial bioprocessing of natural gas and biogas. While recent attempts to engineer synthetic methanol auxotrophs have proved successful, these studies focused on scarce and expensive co-substrates. Here, we engineered E. coli for methanol-dependent growth on glucose, an abundant and inexpensive co-substrate, via deletion of glucose 6-phosphate isomerase (pgi), phosphogluconate dehydratase (edd), and ribose 5-phosphate isomerases (rpiAB). Since the parental strain did not exhibit methanol-dependent growth on glucose in minimal medium, we first achieved methanol-dependent growth via amino acid supplementation and used this medium to evolve the strain for methanol-dependent growth in glucose minimal medium. The evolved strain exhibited a maximum growth rate of 0.15 h−1 in glucose minimal medium with methanol, which is comparable to that of other synthetic methanol auxotrophs. Whole genome sequencing and 13C-metabolic flux analysis revealed the causative mutations in the evolved strain. A mutation in the phosphotransferase system enzyme I gene (ptsI) resulted in a reduced glucose uptake rate to maintain a one-to-one molar ratio of substrate utilization. Deletion of the e14 prophage DNA region resulted in two non-synonymous mutations in the isocitrate dehydrogenase (icd) gene, which reduced TCA cycle carbon flux to maintain the internal redox state. In high cell density glucose fed-batch fermentation, methanol-dependent acetone production resulted in 22% average carbon labeling of acetone from 13C-methanol, which far surpasses that of the previous best (2.4%) found with methylotrophic E. coli Δpgi. This study addresses the need to identify appropriate co-substrates for engineering synthetic methanol auxotrophs and provides a basis for the next steps toward industrial one-carbon bioprocessing.

Phosphate limitation increases coenzyme Q10 production in industrial Rhodobacter sphaeroides HY01

Coenzyme Q10 (CoQ10) is an important component of the respiratory chain in humans and some bacteria. As a high-value-added nutraceutical antioxidant, CoQ10 has excellent capacity to prevent cardiovascular disease. The content of CoQ10 in the industrial Rhodobacter sphaeroides HY01 is hundreds of folds higher than normal physiological levels. In this study, we found that overexpression or optimization of the synthetic pathway failed CoQ10 overproduction in the HY01 strain. Moreover, under phosphate- limited conditions (decreased phosphate or in the absence of inorganic phosphate addition), CoQ10 production increased significantly by 12% to220 mg/L, biomass decreased by 12%, and the CoQ10 productivity of unit cells increased by 27%. In subsequent fed-batch fermentation, CoQ10 production reached 272 mg/L in the shake-flask fermentation and 1.95 g/L in a 100-L bioreactor under phosphate limitation. Furthermore, to understand the mechanism associated with CoQ10 overproduction under phosphate- limited conditions, the comparatve transcriptome analysis was performed. These results indicated that phosphate limitation combined with glucose fed-batch fermentation represented an effective strategy for CoQ10 production in the HY01. Phosphate limitation induced a pleiotropic effect on cell metabolism, and that improved CoQ10 biosynthesis efficiency was possibly related to the disturbance of energy metabolism and redox potential.

Enhanced β-Amyrin Synthesis in Saccharomyces cerevisiae by Coupling An Optimal Acetyl-CoA Supply Pathway.

β-Amyrin is a plant-derived triterpenoid skeleton with wide applications in food and medical industry. β-Amyrin biosynthesis in Saccharomyces cerevisiae is derived from the mevalonate pathway with cytosolic acetyl-CoA as a precursor. In this work, endogenous and several heterologous acetyl-CoA synthesis pathways were coupled to β-amyrin production and a combinational acetyl-CoA supply route was demonstrated to be optimal due to more balanced redox cofactors, much lower energy consumption, and glucose utilization as well as significantly enhanced β-amyrin production (a 200% increase compared to the original β-amyrin-producing strain). Further disruption of an acetyl-CoA competing pathway led to a 330% increase in β-amyrin production as compared to the original strain. Finally, the engineered strain harboring the optimal pathway configuration achieved a final β-amyrin production of 279.0 ± 13.0 mg/L in glucose fed-batch fermentation, which is the highest as ever reported. This work provides an efficient platform for triterpenoid biosynthesis in Saccharomyces cerevisiae.

Overexpression of Malonyl-CoA: ACP Transacylase in Schizochytrium sp. to Improve Polyunsaturated Fatty Acid Production.

Polyunsaturated fatty acids (PUFAs) have been widely applied in the food and medical industry. In this study, malonyl-CoA: ACP transacylase (MAT) was overexpressed through homologous recombination to improve PUFA production in Schizochytrium. The results showed that the lipid and PUFA concentration were increased by 10.1 and 24.5% with MAT overexpression, respectively. Metabolomics analysis revealed that the intracellular tricarboxylic acid cycle was weakened and glucose absorption was accelerated in the engineered strain. In the mevalonate pathway, intracellular carotene content was decreased, and the carbon flux was then redirected toward PUFA synthesis. Furthermore, a glucose fed-batch fermentation was finally performed with the engineered Schizochytrium. The total lipid yield was further increased to 110.5 g/L, 39.6% higher than the wild strain. Docosahexaenoic acid and eicosapentaenoic acid yield were enhanced to 47.39 g/L and 1.65 g/L with an increase of 81.5 and 172.5%, respectively. This study provided an effective metabolic engineering strategy for industrial PUFA production.

Expression of heterologous non-oxidative pentose phosphate pathway from Bacillus methanolicus and phosphoglucose isomerase deletion improves methanol assimilation and metabolite production by a synthetic Escherichia coli methylotroph

Synthetic methylotrophy aims to develop non-native methylotrophic microorganisms to utilize methane or methanol to produce chemicals and biofuels. We report two complimentary strategies to further engineer a previously engineered methylotrophic E. coli strain for improved methanol utilization. First, we demonstrate improved methanol assimilation in the presence of small amounts of yeast extract by expressing the non-oxidative pentose phosphate pathway (PPP) from Bacillus methanolicus. Second, we demonstrate improved co-utilization of methanol and glucose by deleting the phosphoglucose isomerase gene (pgi), which rerouted glucose carbon flux through the oxidative PPP. Both strategies led to significant improvements in methanol assimilation as determined by 13C-labeling in intracellular metabolites. Introduction of an acetone-formation pathway in the pgi-deficient methylotrophic E. coli strain led to improved methanol utilization and acetone titers during glucose fed-batch fermentation.

Improvement of Photorhabdus temperata strain K122 bioinsecticide production by batch and fed-batch fermentations optimization

Optimization of a fermentation process for bioinsecticides production by Photorhabdus temperata strain K122 was investigated into fully controlled 3-L fermenter using an optimized medium (OM). Development of large-scale inocula showed that the composition of the growth medium greatly influenced the physiological state of P. temperata cells. The effect of pH, agitation and dissolved oxygen concentration (DO) on the growth, culturability and oral toxicity of P. temperata cells were also investigated. Indeed, maintaining the pH at 7 and controlling DO concentration at 50 % saturation throughout the fermentation process, improved biomass production, CFU counts and oral toxicity by 41.1, 35 and 32.1 %, respectively, as compared to cultures carried out in 500 mL shake flasks. At such conditions, 8 g/L glucose fed-batch fermentation, enhanced cell lysis and variants small colony (Vsm) polymorphism appearance. To overcome such limitations, glucose concentration should be maintained at 4 g/L. In this case, P. temperata cells were produced at high cell density and culturability reaching 4.5 and 1.2 × 10(9) cells/mL, respectively. In addition, the stability of the primary form was maintained for a long period in the stationary growth phase and Vsm polymorphism was completely avoided that can be crucial for scale-up the bioprocess of P. temperata bioinsecticide.

Fed-batch fermentation and supercritical fluid extraction of heterotrophic microalgal Chlorella protothecoides lipids

Lipids obtained from Chlorella protothecoides in heterotrophic cultivation are considered a suitable feedstock for biodiesel production. In this study, glucose fed-batch fermentation was performed to increase final biomass and lipid production. The biomass productivity and lipid productivity were 6.28 and 2.06g/L day, respectively. Biomass/glucose conversion and the lipid/glucose conversion were 43.3% and 14.2%, respectively. Extraction of lipids from algae has been identified as a key bottleneck in bioprocessing operations. Supercritical carbon dioxide (SC-CO2) was applied for neutral lipids extraction and the SC-CO2 kinetics was investigated by the Goto et al. model. The modeling showed a good fit with experimental data. Additionally, neutral lipids extracted by SC-CO2 displayed a suitable fatty acid profile for biodiesel [mainly C18:1 (60.0%), C18:2 (18.7%) and C16:0 (11.5%)]. Our study demonstrated the ability to produce high levels of neutral lipids through heterotrophic algal culture and subsequent extraction of lipids with SC-CO2 method developed.

Sugar-based growth, astaxanthin accumulation and carotenogenic transcription of heterotrophic Chlorella zofingiensis (Chlorophyta)

The green microalga Chlorella zofingiensis can grow and produce the ketocarotenoid astaxanthin in the dark with glucose as sole carbon and energy source. In the present study, we reported that glucose, mannose, fructose, sucrose, galactose and lactose could differentially support the cell growth and astaxanthin biosynthesis. Of the sugars surveyed, glucose and mannose were the best carbon sources for the algal growth in the dark, as indicated by the relatively high specific growth rates (ca. 0.03 h −1) and high cell dry densities (ca. 10 g l −1). Furthermore, the algal cells cultured with glucose and mannose accumulated the highest amounts of astaxanthin (ca. 1 mg g −1), indicating a correlation between cell growth and astaxanthin formation. In addition, various sugars differentially regulated the transcription of three carotenogenic genes encoding phytoene desaturase (PDS), β-carotene ketolase (BKT), and β-carotene hydroxylase (CHYb) respectively. By using glucose fed-batch fermentation, high cell dry weight concentration (ca. 53 g l −1) and high astaxanthin production (ca. 32 mg l −1) were obtained, which are much higher than those ever reported in the alga. The present study suggests that C. zofingiensis is suitable for the production of natural astaxanthin on a massive scale.