High Cell Density Cultivation Combined with high Specific Enzyme Activity: Cultivation Protocol for the Production of an Amine Transaminase from Bacillus megaterium in E. coli.

High cell density cultivation is an established method for the production of various industrially important products such as recombinant proteins. However, these protocols are not always suitable for biocatalytic processes as the focus often lies on biomass production rather than high specific activities of the enzyme inside the cells. In contrast, a range of shake flask protocols are well known with high specific activities but rather low cell densities. To overcome this gap, we established a tailor-made fed-batch protocol combining both aspects: high cell density and high specific activities of heterologously produced enzyme. Using the example of an industrially relevant amine transaminase from Bacillus megaterium, we describe a strategy to optimize the cultivation yield based on the feed rate, IPTG concentration, and post-induction temperature. By adjusting these key parameters, we were able to increase the specific activity by 2.6-fold and the wet cell weight by even 17-fold compared to shake flasks. Finally, we were able to verify our established protocol by transferring it to another experimenter. With that, our optimization strategy can serve as a template for the production of high titers of heterologously produced, active enzymes and might enable the availability of these catalysts for upscaling biocatalytic processes.

Enhanced poly-γ-glutamic acid synthesis in Corynebacterium glutamicum by reconstituting PgsBCA complex and fermentation optimization.

Previously, a novel Corynebacterium glutamicum strain for the de novo biosynthesis of tailored poly-γ-glutamic acid (γ-PGA) has been constructed by our group. The strain was based on the γ-PGA synthetase complex, PgsBCA, which is the only polyprotein complex responsible for γ-PGA synthesis in Bacillus spp. In the present study, PgsBCA was reconstituted and overexpressed in C. glutamicum to further enhance γ-PGA synthesis. First, we confirmed that all the components (PgsB, PgsC, and PgsA) of γ-PGA synthetase derived from B. licheniformis are necessary for γ-PGA synthesis, and γ-PGA was detected only when PgsB, PgsC, and PgsA were expressed in combination in C. glutamicum. Next, the expression level of each pgsB, pgsC, and pgsA was tuned in order to explore the effect of expression of each of the γ-PGA synthetase subunits on γ-PGA production. Results showed that increasing the transcription levels of pgsB or pgsC and maintaining a medium-level transcription level of pgsA led to 35.44% and 76.53% increase in γ-PGA yield (γ-PGA yield-to-biomass), respectively. Notably, the expression level of pgsC had the greatest influence (accounting for 68.24%) on γ-PGA synthesis, followed by pgsB. Next, genes encoding for PgsC from four different sources (Bacillus subtilis, Bacillus anthracis, Bacillus methylotrophicus, and Bacillus amyloliquefaciens) were tested in order to identify the influence of PgsC-encoding orthologues on γ-PGA production, but results showed that in all cases the synthesis of γ-PGA was significantly inhibited. Similarly, we also explored the influence of gene orthologues encoding for PgsB on γ-PGA production, and found that the titer increased to 17.14 ± 0.62 g/L from 8.24 ± 0.10 g/L when PgsB derived from B. methylotrophicus replaced PgsB alone in PgsBCA from B. licheniformis. The resulting strain was chosen for further optimization, and we achieved a γ-PGA titer of 38.26 g/L in a 5 L fermentor by optimizing dissolved oxygen level. Subsequently, by supplementing glucose, γ-PGA titer increased to 50.2 g/L at 48 h. To the best of our knowledge, this study achieved the highest titer for de novo production of γ-PGA from glucose, without addition of L-glutamic acid, resulting in a novel strategy for enhancing γ-PGA production.

Mass production and characterization of an endoglucanase from Coleoptera insect (Monochamus saltuarius) in yeast Kluyveromyceslactis.

To harness the diverse industrial applications of cellulase, including its use in the food, pulp, textile, agriculture, and biofuel sectors, this study focused on the high-yield production of a bioactive insect-derived endoglucanase, Monochamus saltuarius glycoside hydrolase family 5 (MsGHF5). MsGHF5 was introduced into the genome of Kluyveromyces lactis to maintain expression stability, and mass production of the enzyme was induced using fed-batch fermentation. After 40 h of cultivation, recombinant MsGHF5 was successfully produced in the culture broth, with a yield of 29,000 U/L, upon galactose induction. The optimal conditions for the activity of purified MsGHF5 were determined to be a pH of 5 and a temperature of 35 °C, with the presence of ferrous ions enhancing the enzymatic activity by up to 1.5-fold. Notably, the activity of MsGHF5 produced in K. lactis was significantly higher than that produced in Escherichia coli, suggesting that glycosylation is crucial for the functional performance of the enzyme. This study highlights the potential use of K. lactis as a host for the production of bioactive MsGHF5, thus paving the way for its application in various industrial sectors.

Cytoplasmic production of Fabs in chemically defined media in fed-batch fermentation.

Fragment of antigen-binding region (Fab) of antibodies are important biomolecules, with a broad spectrum of functionality in the biomedical field. While full length antibodies are usually produced in mammalian cells, the smaller size, lack of N-glycosylation and less complex structure of Fabs make production in microbial cell factories feasible. Since Fabs contain disulfide bonds, such production is often done in the periplasm, but there the formation of the inter-molecular disulfide bond between light and heavy chains can be problematic. Here we studied the use of the CyDisCo system (cytoplasmic disulfide bond formation in E. coli) to express two Fabs (Herceptin and Maa48) in the cytoplasm of E. coli in fed-batch fermentation using a generic chemically defined media. We were able to solubly express both Fabs with purified yields of 565 mg/L (Maa48) and 660 mg/L (Herceptin) from low density fermentation. Both proteins exhibited CD spectra consistent with natively folded protein and both were biologically active. To our knowledge this is the first demonstration of high-level production of biological active Fabs in the cytoplasm of E. coli in industrially relevant fermentation conditions.

Thermophilic biocatalysts for one-step conversion of citrus waste into lactic acid.

Agri-food residues offer significant potential as a raw material for the production of L-lactic acid through microbial fermentation. Weizmannia coagulans, previously known as Bacillus coagulans, is a spore-forming, lactic acid-producing, gram-positive, with known probiotic and prebiotic properties. This study aimed to evaluate the feasibility of utilizing untreated citrus waste as a sustainable feedstock for the production of L-lactic acid in a one-step process, by using the strain W. coagulans MA-13. By employing a thermophilic enzymatic cocktail (Cellic CTec2) in conjunction with the hydrolytic capabilities of MA-13, biomass degradation was enhanced by up to 62%. Moreover, batch and fed-batch fermentation experiments demonstrated the complete fermentation of glucose into L-lactic acid, achieving a concentration of up to 44.8 g/L. These results point to MA-13 as a microbial cell factory for one-step production of L-lactic acid, by combining cost-effective saccharification with MA-13 fermentative performance, on agri-food wastes. Moreover, the potential of this approach for sustainable valorization of agricultural waste streams is successfully proven. KEY POINTS: • Valorization of citrus waste, an abundant residue in Mediterranean countries. • Sustainable production of the L-( +)-lactic acid in one-step process. • Enzymatic pretreatment is a valuable alternative to the use of chemical.

Metabolic control analysis enabled the improvement of the L-cysteine production process with Escherichia coli.

L-cysteine is an amino acid with relevance to the pharmaceutical, food, feed, and cosmetic industry. The environmental and societal impact of its chemical production has led to the development of more sustainable fermentative L-cysteine production processes with engineered E. coli based on glucose and thiosulfate as sulphur source. Still, most of the published processes show low yields. For the identification of further metabolic engineering targets, engineered E. coli cells were withdrawn from a fed-batch production process, followed by in vivo metabolic control analysis (MCA) based on the data of short-term perturbation experiments, metabolomics (LC-MS), and thermodynamic flux analysis (TFA). In vivo MCA indicated that the activities of the L-cysteine synthases of the cells withdrawn from the production process might be limiting, and we hypothesised that the L-cysteine precursor O-acetylserine (OAS) might be exported from the cells faster than it took to transform OAS into L-cysteine. By increasing the expression of the L-cysteine synthases, either sulfocysteine synthase or L-cysteine synthase, which transform OAS into L-cysteine, an improvement of up to 70% in specific L-cysteine productivity and up to 47% in the final L-cysteine concentration was achieved in standardised fed-batch processes thereby increasing the yield on glucose by more than 85 to 9.2% (w/w). KEY POINTS: • Metabolic control analysis was applied to analyse L-cysteine production with E. coli • OAS export was faster than its transformation to L-cysteine • Overexpression of L-cysteine synthases improved L-cysteine productivity and yield.

Development of a continuous fermentation process for the production of recombinant uricase enzyme by Pichia pastoris.

Recent studies in the biopharmaceutical industry have shown an increase in the productivity and production efficiency of recombinant proteins by continuous culture. In this research, a new upstream fermentation process was developed for the production of recombinant uricase in the methylotrophic yeast Pichia pastoris. Expression of recombinant protein in this system is under the control of the AOX1 promoter and therefore requires methanol as an inducing agent and carbon/energy source. Considering the biphasic growth characteristics of conventional fed-batch fermentation, physical separation of the growth and induction stages for better control of the continuous fermentation process resulted in higher dry-cell weight (DCW) and enhanced recombinant urate oxidase activity. The DCW and recombinant uricase activity enzyme for fed-batch fermentation were 79 g/L and 6.8 u/mL. During the continuous process, in the growth fermenter at a constant dilution rate of 0.025 h-1 , DCW increased to 88.39 g/L. In the induction fermenter, at methanol feeding rates of 30, 60, and 80 mL/h, a recombinant uricase activity was 4.13, 7.2, and 0 u/mL, respectively. The optimum methanol feeding regime in continuous fermentation resulted in a 4.5-fold improvement in productivity compared with fed-batch fermentation from 0.04 u/mL/h (0.0017 mg/mL/h) to 0.18 u/mL/h (0.0078 mg/mL/h).

Optimization of fermentation conditions for the production of γ-aminobutyric acid by Lactobacillus hilgardii GZ2 from traditional Chinese fermented beverage system.

γ-Aminobutyric acid (GABA) is a crucial neurotransmitter with wide application prospects. In this study, we focused on a GABA-producing strain from a traditional Chinese fermented beverage system. Among the six isolates, Lactobacillus hilgardii GZ2 exhibited the greatest ability to produce GABA in the traditional Chinese fermented beverage system. To increase GABA production, we optimized carbon sources, nitrogen sources, temperature, pH, and monosodium glutamate and glucose concentrations and conducted fed-batch fermentation. The best carbon and nitrogen sources for GABA production and cell growth were glucose, yeast extract and tryptone. Gradual increases in GABA were observed as the glucose and monosodium glutamate concentrations increased from 10 g/L to 50 g/L. During fed-batch fermentation, lactic acid was used to maintain the pH at 5.56, and after feeding with 0.03 g/mL glucose and 0.4 g/mL sodium glutamate for 72 h, the GABA yield reached 239 g/L. This novel high-GABA-producing strain holds great potential for the industrial production of GABA, as well as the development of health-promoting functional foods and medical fields.

Enhancement of ε-poly-L-lysine production by Streptomyces albulus FQF-24 with feeding strategies using cassava starch as carbon source.

ε-Poly-L-lysine (ε-PL) is a natural and wide-spectrum antimicrobial additive. In this study, the production of ε-PL by Streptomyces albulus FQF-24 using cassava starch (CS) as carbon source and the effects of different feeding methods were investigated in a fermenter. The initial shake flask experiments demonstrated the efficient production of ε-PL with CS, achieving the ε-PL production of 1.18 g/L. Subsequent investigations in the fermenter identified that the ideal pH was 3.8 during the ε-PL synthesis phase. Under this condition, the production of ε-PL reached 1.35 g/L. When the pH was maintained at 3.8, the investigation of improvement of feeding composition was carried out in a 5 L fermenter. The intermittent feeding containing CS, inorganic and organic nitrogen sources resulted in the maximum ε-PL production and dry cell weight (DCW) reaching 17.17 g/L and 42.73 g/L. Additionally, continuous feeding with the composition of CS, organic and inorganic nitrogen sources, and inorganic salts further increased ε-PL production and DCW to 27.56 g/L and 38.5 g/L. Summarily, the above results indicate that the fermentation using low-cost CS and continuous feeding strategy with whole medium composition can provide a beneficial reference for the efficient production of ε-PL.

Fed-batch fermentation strategy for efficient welan gum production by Sphingomonas sp. FM01.

BACKGROUND: As a novel type of extracellular polysaccharide produced by Sphingomonas sp., welan gum has been widely applied in various fields because of its excellent properties. The study has improved the fermentation process. RESULTS: The initial sucrose concentration, temperature and stirring speed were set to 20 g L-1, 33 °C and 400 rpm, respectively, and 13.3 g L-1 sucrose was added at 24, 40 and 56 h. The temperature and stirring speed were then set at 28 °C and 600 rpm from 24 to 48 h and 28 °C and 600 rpm from 48 to 72 h, respectively. As a result, welan gum production, dry cell weight, sucrose conversion rate and viscosity were correspondingly increased to 38.60 g L-1, 5.47 g L-1, 0.64 g g-1 and 3779 mPa·s, respectively. In addition, the mechanism by which fermentation strategy promotes welan gum synthesis was investigated by transcriptome analysis. CONCLUSION: Improving respiration and ATP supply, reducing unnecessary protein synthesis, and alleviating competition between cell growth and welan gum synthesis contribute to promoting the fermentation performance of Sphingomonas sp., thus providing a practical strategy for efficient welan gum production. © 2024 Society of Chemical Industry.

Efficient production of polyhydroxybutyrate using lignocellulosic biomass derived from oil palm trunks by the inhibitor-tolerant strain Burkholderia ambifaria E5-3.

Lignocellulosic biomass is a valuable, renewable substrate for the synthesis of polyhydroxybutyrate (PHB), an ecofriendly biopolymer. In this study, bacterial strain E5-3 was isolated from soil in Japan; it was identified as Burkholderia ambifaria strain E5-3 by 16 S rRNA gene sequencing. The strain showed optimal growth at 37 °C with an initial pH of 9. It demonstrated diverse metabolic ability, processing a broad range of carbon substrates, including xylose, glucose, sucrose, glycerol, cellobiose, and, notably, palm oil. Palm oil induced the highest cellular growth, with a PHB content of 65% wt. The strain exhibited inherent tolerance to potential fermentation inhibitors derived from lignocellulosic hydrolysate, withstanding 3 g/L 5-hydroxymethylfurfural and 1.25 g/L acetic acid. Employing a fed-batch fermentation strategy with a combination of glucose, xylose, and cellobiose resulted in PHB production 2.7-times that in traditional batch fermentation. The use of oil palm trunk hydrolysate, without inhibitor pretreatment, in a fed-batch fermentation setup led to significant cell growth with a PHB content of 45% wt, equivalent to 10 g/L. The physicochemical attributes of xylose-derived PHB produced by strain E5-3 included a molecular weight of 722 kDa, a number-average molecular weight of 191 kDa, and a polydispersity index of 3.78. The amorphous structure of this PHB displayed a glass transition temperature of 4.59 °C, while its crystalline counterpart had a melting point of 171.03 °C. This research highlights the potential of lignocellulosic feedstocks, especially oil palm trunk hydrolysate, for PHB production through fed-batch fermentation by B. ambifaria strain E5-3, which has high inhibitor tolerance.

Chromosome evolution of Escherichia coli Nissle 1917 for high-level production of heparosan.

Heparosan is a crucial-polysaccharide precursor for the chemoenzymatic synthesis of heparin, a widely used anticoagulant drug. Presently, heparosan is mainly extracted with the potential risk of contamination from Escherichia coli strain K5, a pathogenic bacterium causing urinary tract infection. Here, a nonpathogenic probiotic, E. coli strain Nissle 1917 (EcN), was metabolically engineered to carry multiple copies of the 19-kb kps locus and produce heparosan to 9.1 g/L in fed-batch fermentation. Chromosome evolution driven by antibiotics was employed to amplify the kps locus, which governed the synthesis and export of heparosan from EcN at 21 mg L-1 OD-1 . The average copy number of kps locus increased from 1 to 24 copies per cell, which produced up to 104 mg L-1 OD-1 of heparosan in the shaking flask cultures of engineered strains. The following in-frame deletion of recA stabilized the recombinant duplicates of chromosomal kps locus and the productivity of heparosan in continuous culture for at least 56 generations. Fed-batch fermentation of the engineered strain EcN8 was carried out to bring the yield of heparosan up to 9.1 g/L. Heparosan from the fermentation culture was further purified at a 75% overall recovery. The structure of purified heparosan was characterized and further modified by N-sulfotransferase with 3'-phosphoadenosine-5'-phosphosulfate as the sulfo-donor. The analysis of element composition showed that heparosan was N-sulfated by over 80%. These results indicated that duplicating large DNA cassettes up to 19-kb, followed by high-cell-density fermentation, was promising in the large-scale preparation of chemicals and could be adapted to engineer other industrial-interest bacteria metabolically.

Optimizing expression of Nanobody® molecules in Pichia pastoris through co-expression of auxiliary proteins under methanol and methanol-free conditions.

BACKGROUND: Ablynx NV, a subsidiary of Sanofi, has a long-standing focus on the development of Nanobody® molecules as biopharmaceuticals (Nanobody® is a registered trademark of Ablynx NV). Nanobody molecules are single variable domains, and they have been met with great success part due to their favorable expression properties in several microbial systems. Nevertheless, the search for the host of the future is an ongoing and challenging process. Komagataella phaffi (Pichia pastoris) is one of the most suitable organisms to produce Nanobody molecules. In addition, genetic engineering of Pichia is easy and an effective approach to improve titers. RESULTS: Here we report that P. pastoris engineered to co-express genes encoding four auxiliary proteins (HAC1, KAR2, PDI and RPP0), leads to a marked improvement in the expression of Nanobody molecules using the AOX1 methanol induction system. Titer improvement is mainly attributed to HAC1, and its beneficial effect was also observed in a methanol-free expression system. CONCLUSION: Our findings are based on over a thousand fed-batch fermentations and offer a valuable guide to produce Nanobody molecules in P. pastoris. The presented differences in expressability between types of Nanobody molecules will be helpful for researchers to select both the type of Nanobody molecule and Pichia strain and may stimulate further the development of a more ecological methanol-free expression platform.

Reduction of acetate synthesis, enhanced arginine export, and supply of precursors, cofactors, and energy for improved synthesis of L-arginine by Escherichia coli.

L-arginine (L-Arg) is a semi-essential amino acid with many important physiological functions. However, achieving efficient manufacture of L-Arg on an industrial scale using Escherichia coli (E. coli) remains a major challenge. In previous studies, we constructed a strain of E. coli A7, which had good L-Arg production capacity. In this study, E. coli A7 was further modified, and E. coli A21 with more efficient L-Arg production capacity was obtained. Firstly, we reduced the acetate accumulation of strain A7 by weakening the poxB gene and overexpressing acs gene. Secondly, we improved the L-Arg transport efficiency of strains by overexpressing the lysE gene from Corynebacterium glutamicum (C. glutamicum). Finally, we enhanced the supplies of precursors for the synthesis of L-Arg and optimized the supplies of cofactor NADPH and energy ATP in strain. After fermentation in a 5-L bioreactor, the L-Arg titer of strain A21 was found to be 89.7 g/L. The productivity was 1.495 g/(L·h) and the glucose yield was 0.377 g/g. Our study further narrowed the titer gap between E. coli and C. glutamicum in the synthesis of L-Arg. In all recent studies on the L-Arg production by E. coli, this was the highest titer recorded. In conclusion, our study further promotes the efficient mass synthesis of L-Arg by E. coli. KEY POINTS: • The acetate accumulation of starting strain A7 was decreased. • Overexpression of gene lysE of C. glutamicum enhanced L-Arg transport in strain A10. • Enhance the supplies of precursors for the synthesis of L-Arg and optimize the supplies of cofactor NADPH and energy ATP. Finally, Strain A21 was detected to have an L-Arg titer of 89.7 g/L in a 5-L bioreactor.

Development of probiotic E. coli Nissle 1917 for ß-alanine production by using protein and metabolic engineering.

ß-alanine has been used in food and pharmaceutical industries. Although Escherichia coli Nissle 1917 (EcN) is generally considered safe and engineered as living therapeutics, engineering EcN for producing industrial metabolites has rarely been explored. Here, by protein and metabolic engineering, EcN was engineered for producing ß-alanine from glucose. First, an aspartate-α-decarboxylase variant ADCK43Y with improved activity was identified and over-expressed in EcN, promoting the titer of ß-alanine from an undetectable level to 0.46 g/L. Second, directing the metabolic flux towards L-aspartate increased the titer of ß-alanine to 0.92 g/L. Third, the yield of ß-alanine was elevated to 1.19 g/L by blocking conversion of phosphoenolpyruvate to pyruvate, and further increased to 2.14 g/L through optimizing culture medium. Finally, the engineered EcN produced 11.9 g/L ß-alanine in fed-batch fermentation. Our work not only shows the potential of EcN as a valuable industrial platform, but also facilitates production of ß-alanine via fermentation. KEY POINTS: • Escherichia coli Nissle 1917 (EcN) was engineered as a ß-alanine producing cell factory • Identification of a decarboxylase variant ADCK43Y with improved activity • Directing the metabolic flux to L-ASP and expressing ADCK43Y elevated the titer of ß-alanine to 11.9 g/L.

Methanol bioconversion in Methylotuvimicrobium buryatense 5GB1C through self-cycling fermentation.

Methanol is an abundant and low-cost next-generation carbon source. While many species of methanotrophic bacteria can convert methanol into valuable bioproducts in bioreactors, Methylotuvimicrobium buryatense 5GB1C stands out as one of the most promising strains for industrialization. It has a short doubling time compared to most methanotrophs, remarkable resilience against contamination, and a suite of tools enabling genetic engineering. When approaching industrial applications, growing M. buryatense 5GB1C on methanol using common batch reactor operation has important limitations; for example methanol toxicity leads to mediocre biomass productivity. Advanced bioreactor operation strategies, such as fed-batch and self-cycling fermentation, have the potential to greatly improve the industrial prospects of methanotrophs growing on methanol. Herein, implementation of fed-batch operation led to a 26-fold increase in biomass density, while two different self-cycling fermentation (SCF) strategies led to 3-fold and 10-fold increases in volumetric biomass productivity. Interestingly, while synchronization is a typical trait of microbial populations undergoing SCF, M. buryatense 5GB1C cultures growing under this mode of operation led to stable, reproducible cycles but no significant synchronization.

Enhancement of acarbose production by genetic engineering and fed-batch fermentation strategy in Actinoplanes sp. SIPI12-34.

BACKGROUND: Acarbose, as an alpha-glucosidase inhibitor, is widely used clinically to treat type II diabetes. In its industrial production, Actinoplanes sp. SE50/110 is used as the production strain. Lack of research on its regulatory mechanisms and unexplored gene targets are major obstacles to rational strain design. Here, transcriptome sequencing was applied to uncover more gene targets and rational genetic engineering was performed to increase acarbose production. RESULTS: In this study, with the help of transcriptome information, a TetR family regulator (TetR1) was identified and confirmed to have a positive effect on the synthesis of acarbose by promoting the expression of acbB and acbD. Some genes with low expression levels in the acarbose biosynthesis gene cluster were overexpressed and this resulted in a significant increase in acarbose yield. In addition, the regulation of metabolic pathways was performed to retain more glucose-1-phosphate for acarbose synthesis by weakening the glycogen synthesis pathway and strengthening the glycogen degradation pathway. Eventually, with a combination of multiple strategies and fed-batch fermentation, the yield of acarbose in the engineered strain increased 58% compared to the parent strain, reaching 8.04 g/L, which is the highest fermentation titer reported. CONCLUSIONS: In our research, acarbose production had been effectively and steadily improved through genetic engineering based on transcriptome analysis and fed-batch culture strategy.

Cell factory for γ-aminobutyric acid (GABA) production using Bifidobacterium adolescentis.

BACKGROUND: Bifidobacteria are gram-positive, probiotic, and generally regarded as safe bacteria. Techniques such as transformation, gene knockout, and heterologous gene expression have been established for Bifidobacterium, indicating that this bacterium can be used as a cell factory platform. However, there are limited previous reports in this field, likely because of factors such as the highly anaerobic nature of this bacterium. Bifidobacterium adolescentis is among the most oxygen-sensitive Bifidobacterium species. It shows strain-specific gamma-aminobutyric acid (GABA) production. GABA is a potent bioactive compound with numerous physiological and psychological functions. In this study, we investigated whether B. adolesentis could be used for mass production of GABA. RESULTS: The B. adolescentis 4-2 strain isolated from a healthy adult human produced approximately 14 mM GABA. It carried gadB and gadC, which encode glutamate decarboxylase and glutamate GABA antiporter, respectively. We constructed pKKT427::Pori-gadBC and pKKT427::Pgap-gadBC plasmids carrying gadBC driven by the original gadB (ori) and gap promoters, respectively. Recombinants of Bifidobacterium were then constructed. Two recombinants with high production abilities, monitored by two different promoters, were investigated. GABA production was improved by adjusting the fermentation parameters, including the substrate concentration, initial culture pH, and co-factor supplementation, using response surface methodology. The optimum initial cultivation pH varied when the promoter region was changed. The ori promoter was induced under acidic conditions (pH 5.2:4.4), whereas the constitutive gap promoter showed enhanced GABA production at pH 6.0. Fed-batch fermentation was used to validate the optimum fermentation parameters, in which approximately 415 mM GABA was produced. The conversion ratio of glutamate to GABA was 92-100%. CONCLUSION: We report high GABA production in recombinant B. adolescentis. This study provides a foundation for using Bifidobacterium as a cell factory platform for industrial production of GABA.

Effect of levulinic acid on production of polyhydroxyalkanoates from food waste by Haloferax mediterranei.

Polyhydroxyalkanoates (PHA), especially poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) is considered as the most suitable candidate to replace petrochemical plastics. However, the high production cost and the composition of the monomers in the copolymer are the major constraints in production. The 3-hydroxyvalerate (3HV) rich copolymers are ideal for various applications due to their lower melting points, improved elasticity, and ductility. Haloferax mediterranei is a suitable microorganism for the production of biopolymer PHBV from biowaste. Nevertheless, the potential of H. mediterranei cultivated on food waste as sustainable substrate and levulinic acid as an inducer has not been explored for PHBV production. This study aims at the valorization of food waste as low-cost substrate and evaluation of effect of levulinic acid in the production and composition of PHBV using H. mediterranei. Shake-flask fermentations using different concentrations of salt, glucose and levulinic acid were first performed to optimize the cultivation conditions. The highest growth of the halophile was observed at salt concentration of 15% and glucose of concentration 10 g/L. Under optimized growth conditions, H. mediterranei was cultivated for PHBV production in fed-batch bioreactor with pulse fed levulinic acid. The maximum biomass of 3.19 ± 0.66 g/L was achieved after 140 h of cultivation with 3 g/L of levulinic acid. A decrease in H. mediterranei growth was noticed with the increase in levulinic acid concentration in the range of 3-10 g/L. The overall yield of PHBV at 3, 5, 7 and 10 g/L of levulinic acid were 18.23%, 56.70%, 31.54%, 21.29%, respectively. The optimum concentration of 5 g/L of levulinic acid was found to produce the maximum yield of 56.70% PHBV with 18.55 mol% 3HV content. A correlation between levulinic acid concentrations and PHBV production established in this study can serve as an important reference for future large-scale production.

Pilot-scale production of exopolysaccharide from Leuconostoc pseudomesenteroides XG5 and its application in set yogurt.

Exopolysaccharide from Leuconostoc pseudomesenteroides XG5 (XG5 EPS) is a linear dextran that is built by glucose units via α-1,6 glycosidic bond. The primary objective of this study was to investigate the yield of XG5 EPS and its application in set yogurt. In laboratory scale, the culture conditions of XG5 EPS production were optimized using the L9 (33) orthogonal test. Here, the optimized yield of XG5 EPS was 26.02 g/L under the conditions of 100 g/L sucrose, initial pH 7.0, 25°C incubation, and 100 rpm for 36 h in a shaking flask. Based on the optimized parameters of laboratory scale, a pilot fed-batch fermentation was performed in a 50-L bioreactor with an adjusted agitation speed of 20 rpm. The XG5 EPS yield reached 40.07 g/L in fed-batch fermentation, which was 54% higher than that achieved in laboratory scale. In addition, XG5 EPS was added into set yogurt to investigate its effect on the stability of set yogurt. Our data demonstrated that the XG5 EPS improved the water-holding capacity, texture profile, and viscosity of set yogurt during cold storage compared with the controls. In particular, addition of 0.5% XG5 EPS increased the structure of 3-dimensional network of set yogurt, which eventually improved the physical stability of the set yogurt. Overall, this study provided new insights for exploring the pilot scale production and application of dextran.