Enhanced fed-batch production of pyrroloquinoline quinine in <Emphasis Type="Italic">Methylobacillus</Emphasis> sp. CCTCC M2016079 with a two-stage pH control strategy

The effects of pH control strategy and fermentative operation modes on the biosynthesis of pyrroloquinoline quinine (PQQ) were investigated systematically with Methylobacillus sp. CCTCC M2016079 in the present work. Firstly, the shake-flask cultivations and benchtop fermentations at various pH values ranging from 5.3 to 7.8 were studied. Following a kinetic analysis of specific cell growth rate (μ _x ) and specific PQQ formation rate (μ _p ), the discrepancy in optimal pH values between cell growth and PQQ biosynthesis was observed, which stimulated us to develop a novel two-stage pH control strategy. During this pH-shifted process, the pH in the broth was controlled at 6.8 to promote the cell growth for the first 48 h and then shifted to 5.8 to enhance the PQQ synthesis until the end of fermentation. By applying this pH-shifted control strategy, the maximum PQQ production was improved to 158.61 mg/L in the benchtop fermenter, about 44.9% higher than that under the most suitable constant pH fermentation. Further fed-batch study showed that PQQ production could be improved from 183.38 to 272.21 mg/L by feeding of methanol at the rate of 11.5 mL/h in this two-stage pH process. Meanwhile, the productivity was also increased from 2.02 to 2.84 mg/L/h. In order to support cell growth during the shifted pH stage, the combined feeding of methanol and yeast extract was carried out, which brought about the highest concentration (353.28 mg/L) and productivity (3.27 mg/L/h) of PQQ. This work has revealed the potential of our developed simple and economical strategy for the large-scale production of PQQ.     

Valorization of Hemicelluloses: Production of Bioxylitol from Poplar Wood Prehydrolyzates by <Emphasis Type="Italic">Candida guilliermondii</Emphasis> FTI 20037

In this study on the valorization of hemicelluloses (a co-product generated during cellulosic bioethanol production), prehydrolyzates obtained from poplar woodchips pretreated in an industrial experimental steam-explosion pilot-plant facility were evaluated for the production of bioxylitol using the yeast, Candida guilliermondii FTI 20037, employing both batch and fed-batch fermentation modes in shake flasks on defined nutrient medium. The prehydrolyzates consisted of monosaccharides (pentose and hexose sugars) as well as xylo-oligosaccharides and undegraded hemicellulose. Xylose (31.6?±?0.57 g/L) was the major sugar in the prehydrolyzates that also contained acetic acid and degradation products of lignin and sugars (phenolic and furanic compounds). Xylose in the prehydrolyzates could be further increased (106.4?±?0.02 g/L) through an acid hydrolysis step (0.6 % (w/v) H₂SO₄). Compounds of a toxic nature in both the acid hydrolyzates and prehydrolyzates were removed by treatment with Amberlite IRA-400 resin (chloride form). Batch fermentation of pure xylose and poplar prehydrolyzate resulted in bioxylitol production of 9.9?±?0.01 and 4.9?±?0.17 g/L, respectively, indicating that the poplar prehydrolyzates exhibited an inhibitory effect on fermentation. After detoxification of the poplar prehydrolyzates, bioxylitol production increased to 8.9?±?0.01 g/L. Fed-batch fermentation of the prehydrolyzate increased the bioxylitol production to 12.39?±?0.33 g/L, while acid hydrolysis followed by detoxification resulted in a maximum bioxylitol production of 22.0?±?0.01 g/L, a 348 % increase. The results demonstrated that acid hydrolysis and detoxification followed by fed-batch fermentation was an efficient way to produce bioxylitol from poplar prehydrolyzates.     

Interaction of <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>–<Emphasis Type="Italic">Lactobacillus fermentum</Emphasis>–<Emphasis Type="Italic">Dekkera bruxellensis</Emphasis> and feedstock on fuel ethanol fermentation

The alcoholic fermentation for fuel ethanol production in Brazil occurs in the presence of several microorganisms present with the starter strain of Saccharomyces cerevisiae in sugarcane musts. It is expected that a multitude of microbial interactions may exist and impact on the fermentation yield. The yeast Dekkera bruxellensis and the bacterium Lactobacillus fermentum are important and frequent contaminants of industrial processes, although reports on the effects of both microorganisms simultaneously in ethanolic fermentation are scarce. The aim of this work was to determine the effects and interactions of both contaminants on the ethanolic fermentation carried out by the industrial yeast S. cerevisiae PE-2 in two different feedstocks (sugarcane juice and molasses) by running multiple batch fermentations with the starter yeast in pure or co-cultures with D. bruxellensis and/or L. fermentum. The fermentations contaminated with D. bruxellensis or L. fermentum or both together resulted in a lower average yield of ethanol, but it was higher in molasses than that of sugarcane juice. The decrease in the CFU number of S. cerevisiae was verified only in co-cultures with both D. bruxellensis and L. fermentum concomitant with higher residual sucrose concentration, lower glycerol and organic acid production in spite of a high reduction in the medium pH in both feedstocks. The growth of D. bruxellensis was stimulated in the presence of L. fermentum resulting in a more pronounced effect on the fermentation parameters than the effects of contamination by each microorganism individually.     

Enhanced production of 2,3-butanediol by engineered <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> through fine-tuning of pyruvate decarboxylase and NADH oxidase activities

Background

2,3-Butanediol (2,3-BD) is a promising compound for various applications in chemical, cosmetic, and agricultural industries. Pyruvate decarboxylase (Pdc)-deficient Saccharomyces cerevisiae is an attractive host strain for producing 2,3-BD because a large amount of pyruvate could be shunted to 2,3-BD production instead of ethanol synthesis. However, 2,3-BD yield, productivity, and titer by engineered yeast were inferior to native bacterial producers because of the following metabolic limitations. First, the Pdc-deficient yeast showed growth defect due to a shortage of C₂-compounds. Second, redox imbalance during the 2,3-BD production led to glycerol formation that lowered the yield.

Results

To overcome these problems, the expression levels of Pdc from a Crabtree-negative yeast were optimized in S. cerevisiae. Specifically, Candida tropicalis PDC1 (CtPDC1) was used to minimize the production of ethanol but maximize cell growth and 2,3-BD productivity. As a result, productivity of the BD5_G1CtPDC1 strain expressing an optimal level of Pdc was 2.3 folds higher than that of the control strain in flask cultivation. Through a fed-batch fermentation, 121.8 g/L 2,3-BD was produced in 80 h. NADH oxidase from Lactococcus lactis (noxE) was additionally expressed in the engineered yeast with an optimal activity of Pdc. The fed-batch fermentation with the optimized 2-stage aeration control led to production of 154.3 g/L 2,3-BD in 78 h. The overall yield of 2,3-BD was 0.404 g 2,3-BD/g glucose which corresponds to 80.7% of theoretical yield.

Conclusions

A massive metabolic shift in the engineered S. cerevisiae (BD5_G1CtPDC1_nox) expressing NADH oxidase was observed, suggesting that redox imbalance was a major bottleneck for efficient production of 2,3-BD by engineered yeast. Maximum 2,3-BD titer in this study was close to the highest among the reported microbial production studies. The results demonstrate that resolving both C₂-compound limitation and redox imbalance is critical to increase 2,3-BD production in the Pdc-deficient S. cerevisiae. Our strategy to express fine-tuned PDC and noxE could be applicable not only to 2,3-BD production, but also other chemical production systems using Pdc-deficient S. cerevisiae.

     

Enhancing carotenoid production in <Emphasis Type="Italic">Rhodotorula mucilaginosa</Emphasis> KC8 by combining mutation and metabolic engineering

Rhodotorula mucilaginosa has been considered as a potential industrial yeast due to its unicellular and fast-growing characteristics, and its ability to produce carotenoids, including torularhodin. However, its low total carotenoid production limits its commercial application. In this study, mutation breeding and metabolic engineering were employed to enhance carotenoid production in the R. mucilaginosa strain KC8. After chemical–physical mutagenesis, R. mucilaginosa K4 with a 67% greater concentration of carotenoids (14.47 ± 0.06 mg L^?1) than R. mucilaginosa KC8 (8.67 ± 0.07 mg L^?1) was obtained. To further enhance carotenoid production, gene HMG1 encoding the 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase was introduced from another yeast, Saccharomyces cerevisiae, and overexpressed in R. mucilaginosa K4. The carotenoid production of HMG1-gene-overexpression transformant G1 reached 16.98 mg L^?1. To relieve the feedback inhibition of ergosterol, and to down-regulate ergosterol synthesis, ketoconazole, an ergosterol synthesis inhibitor, was added at a concentration of 28 mg L^?1. The carotenoid production of the transformant G1 reached 19.14 ± 0.09 mg L^?1, which was 121% higher than in R. mucilaginosa KC8. This suggests that a combination of chemical–physical mutagenesis, overexpression of the HMG1 gene, and adding ketoconazole is an effective strategy to improve carotenoid production.     

Enhanced isopropanol and <Emphasis Type="Italic">n</Emphasis>-butanol production by supplying exogenous acetic acid via co-culturing two clostridium strains from cassava bagasse hydrolysate

The focus of this study was to produce isopropanol and butanol (IB) from dilute sulfuric acid treated cassava bagasse hydrolysate (SACBH), and improve IB production by co-culturing Clostridium beijerinckii (C. beijerinckii) with Clostridium tyrobutyricum (C. tyrobutyricum) in an immobilized-cell fermentation system. Concentrated SACBH could be converted to solvents efficiently by immobilized pure culture of C. beijerinckii. Considerable solvent concentrations of 6.19 g/L isopropanol and 12.32 g/L butanol were obtained from batch fermentation, and the total solvent yield and volumetric productivity were 0.42 g/g and 0.30 g/L/h, respectively. Furthermore, the concentrations of isopropanol and butanol increased to 7.63 and 13.26 g/L, respectively, under the immobilized co-culture conditions when concentrated SACBH was used as the carbon source. The concentrations of isopropanol and butanol from the immobilized co-culture fermentation were, respectively, 42.62 and 25.45 % higher than the production resulting from pure culture fermentation. The total solvent yield and volumetric productivity increased to 0.51 g/g and 0.44 g/L/h when co-culture conditions were utilized. Our results indicated that SACBH could be used as an economically favorable carbon source or substrate for IB production using immobilized fermentation. Additionally, IB production could be significantly improved by co-culture immobilization, which provides extracellular acetic acid to C. beijerinckii from C. tyrobutyricum. This study provided a technically feasible and cost-efficient way for IB production using cassava bagasse, which may be suitable for industrial solvent production.     

<Emphasis Type="Italic">R</Emphasis>-acetoin accumulation and dissimilation in <Emphasis Type="Italic">Klebsiella pneumoniae</Emphasis>

Klebsiella pneumoniae is a 2,3-butanediol producer, and R-acetoin is an intermediate of 2,3-butanediol production. R-acetoin accumulation and dissimilation in K. pneumoniae was studied here. A budC mutant, which has lost 2,3-butanediol dehydrogenase activity, accumulated high levels of R-acetoin in culture broth. However, after glucose was exhausted, the accumulated R-acetoin could be reused by the cells as a carbon source. Acetoin dehydrogenase enzyme system, encoded by acoABCD, was responsible for R-acetoin dissimilation. acoABCD mutants lost the ability to grow on acetoin as the sole carbon source, and the acetoin accumulated could not be dissimilated. However, in the presence of another carbon source, the acetoin accumulated in broth of acoABCD mutants was converted to 2,3-butanediol. Parameters of R-acetoin production by budC mutants were optimized in batch culture. Aerobic culture and mildly acidic conditions (pH 6–6.5) favored R-acetoin accumulation. At the optimized conditions, in fed-batch fermentation, 62.3 g/L R-acetoin was produced by budC and acoABCD double mutant in 57 h culture, with an optical purity of 98.0 %, and a substrate conversion ratio of 28.7 %.     

Plant growth-promoting traits of epiphytic and endophytic yeasts isolated from rice and sugar cane leaves in Thailand

A total of 1035 yeast isolates, obtained from rice and sugar cane leaves, were screened primarily for indole-3-acetic acid (IAA) production. Thirteen isolates were selected, due to their IAA production ranging from 1.2 to 29.3 mg g⁻¹ DCW. These isolates were investigated for their capabilities of calcium phosphate and ZnO₃ solubilisation, and also for production of NH₃, polyamine, and siderophore. Their 1-aminocyclopropane-1-carboxylate (ACC) deaminase, catalase and fungal cell wall-degrading enzyme activities were assessed. Their antagonism against rice fungal pathogens was also evaluated. Strain identification, based on molecular taxonomy, of the thirteen yeast isolates revealed that four yeast species – i.e. Hannaella sinensis (DMKU-RP45), Cryptococcus flavus (DMKU-RE12, DMKU-RE19, DMKU-RE67, and DMKU-RP128), Rhodosporidium paludigenum (DMKU-RP301) and Torulaspora globosa (DMKU-RP31) – were capable of high IAA production. Catalase activity was detected in all yeast strains tested. The yeast R. paludigenum DMKU-RP301 was the best IAA producer, yielding 29.3 mg g⁻¹ DCW, and showed the ability to produce NH₃ and siderophore. Different levels of IAA production (7.2–9.7 mg g⁻¹ DCW) were found in four strains of C. flavus DMKU-RE12, DMKU-RE19, and DMKU-RE67, which are rice leaf endophytes, and strain DMKU-RP128, which is a rice leaf epiphyte. NH₃ production and carboxymethyl cellulase (CMCase) activity was also detected in these four strains. Antagonism to fungal plant pathogens and production of antifungal volatile compounds were exhibited in T. globosa DMKU-RP31, as well as a moderate level of IAA production (4.9 mg g⁻¹ DCW). The overall results indicated that T. globosa DMKU-RP31 might be used in two ways: enhancing plant growth and acting as a biocontrol agent. In addition, four C. flavus were also found to be strains of interest for optimal IAA production.     

Production of optically pure 2,3-butanediol from <Emphasis Type="Italic">Miscanthus floridulus</Emphasis> hydrolysate using engineered <Emphasis Type="Italic">Bacillus licheniformis</Emphasis> strains

2,3-Butanediol (2,3-BD) can be produced by fermentation of natural resources like Miscanthus. Bacillus licheniformis mutants, WX-02ΔbudC and WX-02ΔgldA, were elucidated for the potential to use Miscanthus as a cost-effective biomass to produce optically pure 2,3-BD. Both WX-02ΔbudC and WX-02ΔgldA could efficiently use xylose as well as mixed sugars of glucose and xylose to produce optically pure 2,3-BD. Batch fermentation of M. floridulus hydrolysate could produce 21.6 g/L d-2,3-BD and 23.9 g/L meso-2,3-BD in flask, and 13.8 g/L d-2,3-BD and 13.2 g/L meso-2,3-BD in bioreactor for WX-02ΔbudC and WX-02ΔgldA, respectively. Further fed-batch fermentation of hydrolysate in bioreactor showed both of two strains could produce optically pure 2,3-BD, with 32.2 g/L d-2,3-BD for WX-02ΔbudC and 48.5 g/L meso-2,3-BD for WX-02ΔgldA, respectively. Collectively, WX-02ΔbudC and WX-02ΔgldA can efficiently produce optically pure 2,3-BD with M. floridulus hydrolysate, and these two strains are candidates for industrial production of optical purity of 2,3-BD with M. floridulus hydrolysate.     

Deletion of <Emphasis Type="Italic">ldh</Emphasis>A and <Emphasis Type="Italic">ald</Emphasis>H genes in <Emphasis Type="Italic">Klebsiella</Emphasis><Emphasis Type="Italic">pneumoniae</Emphasis> to enhance 1,3-propanediol production

Objectives

To improve 1,3-propanediol (1,3-PD) production and reduce byproduct concentration during the fermentation of Klebsiella pneumonia.

Results

Klebsiella. pneumonia 2-1ΔldhA, K. pneumonia 2-1ΔaldH and K. pneumonia 2-1ΔldhAΔaldH mutant strains were obtained through deletion of the ldhA gene encoding lactate dehydrogenase required for lactate synthesis and the aldH gene encoding acetaldehyde dehydrogenase involved in the synthesis of ethanol. After fed-batch fermentation, the production of 1,3-PD from glycerol was enhanced and the concentrations of byproducts were reduced compared with the original strain K. pneumonia 2-1. The maximum yields of 1,3-PD were 85.7, 82.5 and 87.5 g/l in the respective mutant strains.

Conclusion

Deletion of either aldH or ldhA promoted 1,3-PD production in K. pneumonia.

     

Production of carotenoids and lipids by <Emphasis Type="Italic">Rhodococcus opacus</Emphasis> PD630 in batch and fed-batch culture

Production of carotenoids by Rhodococcus opacus PD630 is reported. A modified mineral salt medium formulated with glycerol as an inexpensive carbon source was used for the fermentation. Ammonium acetate was the nitrogen source. A dry cell mass concentration of nearly 5.4 g/L could be produced in shake flasks with a carotenoid concentration of 0.54 mg/L. In batch culture in a 5 L bioreactor, without pH control, the maximum dry biomass concentration was ~30 % lower than in shake flasks and the carotenoids concentration was 0.09 mg/L. Both the biomass concentration and the carotenoids concentration could be raised using a fed-batch operation with a feed mixture of ammonium acetate and acetic acid. With this strategy, the final biomass concentration was 8.2 g/L and the carotenoids concentration was 0.20 mg/L in a 10-day fermentation. A control of pH proved to be unnecessary for maximizing the production of carotenoids in this fermentation.     

Yeasts from peat in a tropical peat swamp forest in Thailand and their ability to produce ethanol,indole-3-acetic acid and extracellular enzymes

This study aimed to isolate and identify yeasts from peat in To Daeng peat swamp forest in southern of Thailand, and to investigate their ability to produce ethanol from glucose and xylose and to produce indole-3-acetic acid (IAA) and extracellular enzymes. A total of 65 yeast strains were obtained from 15 peat samples using an enrichment technique, and 61 strains were identified to be five species belonging to the phylum Ascomycota, namely Cyberlindnera subsufficiens, Debaryomyces fabryi, Meyerozyma guilliermondii, Saturnispora diversa and Schwanniomyces polymorphus var. africanus, and five species of the phylum Basidiomycota, namely Cryptococcus taiwanensis pro tem, Cutaneotrichosporon mucoides, Papiliotrema flavescens, Papiliotrema laurentii and Rhodotorula mucilaginosa. Four strains were unidentified and require further analysis. They differed from the type strain of P. flavescens by two nucleotide substitutions in the D1/D2 region of the LSU rRNA gene and nine nucleotide substitutions in the ITS region. R. mucilaginosa was the most prevalent yeast species, followed by S. polymorphus var. africanus, Cy. subsufficiens and D. fabryi. None of the yeast strains obtained in this study were able to ferment xylose to ethanol, but all ascomycetous yeast strains produced ethanol from glucose in a range of 9.0–58.0 g/L, with Cy. subsufficiens DMKU-YNB42-1 producing the highest ethanol concentration. A total of 62 strains produced IAA in a range of 9.0 to 66.9 mg/L, with the highest IAA produced by R. mucilaginosa DMKU-Y33-A. Investigation of the production of cellulases, xylanase, pectinase, amylase, protease and lipase revealed that all 65 yeast strains produced at least one extracellular enzyme, a lipase.     

An optimized fed-batch culture strategy integrated with a one-step fermentation improves <Emphasis Type="SmallCaps">l</Emphasis>-lactic acid production by <Emphasis Type="Italic">Rhizopus oryzae</Emphasis>

In previous work, we proposed a novel modified one-step fermentation fed-batch strategy to efficiently generate l-lactic acid (l-LA) using Rhizopus oryzae. In this study, to further enhance efficiency of l-LA production through one-step fermentation in fed-batch cultures, we systematically investigated the initial peptone- and glucose-feeding approaches, including different initial peptone and glucose concentrations and maintained residual glucose levels. Based on the results of this study, culturing R. oryzae with initial peptone and glucose concentrations of 3.0 and 50.0 g/l, respectively, using a fed-batch strategy is an effective approach of producing l-LA through one-step fermentation. Changing the residual glucose had no obvious effect on the generation of l-LA. We determined the maximum LA production and productivity to be 162 g/l and 6.23 g/(l·h), respectively, during the acid production stage. Compared to our previous work, there was almost no change in l-LA production or yield; however, the productivity of l-LA increased by 14.3%.     

Enhanced truncated-t-PA (CT-b) expression in high-cell-density fed-batch cultures of <Emphasis Type="Italic">Pichia pastoris</Emphasis> through optimization of a mixed feeding strategy by response surface methodology

Recently, Pichia pastoris has been the focal point of interest as an expression system for production of many recombinant proteins. The study and optimization of feeding strategy are of major importance to achieve maximum volumetric productivity in fed-batch cultivations. Among different feeding strategies used in P. pastoris fed-batch cultures, those trying to maintain a constant specific growth rate have usually resulted in superior productivities. The objective of the present study was to investigate and optimize the co-feeding of glycerol and methanol to attain maximum expression of t-PA in P. pastoris fed-batch cultures with constant specific growth rate. The experiments were designed by response surface methodology, considering the specific feeding rates of methanol and glycerol as independent variables. In each experiment, glycerol and methanol were fed according to a predetermined equation to maintain a constant specific growth rate. It was found that with glycerol feeding for higher specific growth rates, the inhibitory properties of glycerol are more pronounced, while the best expression level was achieved when the ratio of µ _set glycerol to that of methanol was around 1.67. In all specific growth rates tested, almost a similar ratio of the specific glycerol feeding rate to that of methanol led to the maximum protein production and activity. The statistical model predicted the optimal operating conditions for µ _set glycerol and that of methanol to be 0.05 and 0.03 h^?1, respectively. Applying the optimum strategy, maximum of 52 g/L biomass, 300 mg/L t-PA and 340,000 IU/mL enzyme activity were obtained.     

Engineering of the carotenoid pathway in <Emphasis Type="Italic">Xanthophyllomyces dendrorhous</Emphasis> leading to the synthesis of zeaxanthin

Zeaxanthin is an essential nutrient for prevention of macular degeneration. However, it is limited in our diet. For the production of zeaxanthin, we have engineered zeaxanthin synthesis into a carotenoid mutant of Xanthophyllomyces dendrorhous which is blocked in astaxanthin synthesis and accumulates β-carotene instead. Two strategies were followed to reach high-yield zeaxanthin synthesis. Total carotenoid synthesis was increased by over-expression of genes HMGR, crtE, and crtYB encoding for limiting enzymes in the pathway leading to and into carotenoid biosynthesis. Then bacterial genes crtZ were used to extend the pathway from β-carotene to zeaxanthin in this mutant. The increase of total carotenoids and the formation of zeaxanthin is dependent on the number of gene copies of crtYB and crtZ integrated into the X. dendrorhous upon transformation. The highest zeaxanthin content around 500 μg/g dw was reached by shaking flask cultures after codon optimization of crtZ for Xanthophyllomyces. Stabilization of carotenoid and zeaxanthin formation in the final transformant in the absence of selection agents was achieved after passing through a sexual cycle and germination of basidiospores. The values for the transformant before and after stabilization were very similar resembling about 70 % of total carotenoids and corresponding to a conversion rate of 80 % for hydroxylation of β-carotene to zeaxanthin. The stabilized transformant allowed experimental small-scale fermentation yielding X. dendrorhous cells with a zeaxanthin content similar to the shaking flask cultures. Our result demonstrates the potential of X. dendrorhous for its development as a zeaxanthin producer and its suitability for large-scale fermentation.     

Rational design and analysis of an <Emphasis Type="Italic">Escherichia coli</Emphasis> strain for high-efficiency tryptophan production

l-tryptophan (l-trp) is a precursor of various bioactive components and has great pharmaceutical interest. However, due to the requirement of several precursors and complex regulation of the pathways involved, the development of an efficient l-trp production strain is challenging. In this study, Escherichia coli (E. coli) strain KW001 was designed to overexpress the l-trp operator sequences (trpEDCBA) and 3-deoxy-D-arabinoheptulosonate-7-phosphate synthase (aroG ^fbr ). To further improve the production of l-trp, pyruvate kinase (pykF) and the phosphotransferase system HPr (ptsH) were deleted after inactivation of repression (trpR) and attenuation (attenuator) to produce strain KW006. To overcome the relatively slow growth and to increase the transport rate of glucose, strain KW018 was generated by combinatorial regulation of glucokinase (galP) and galactose permease (glk) expression. To reduce the production of acetic acid, strain KW023 was created by repressive regulation of phosphate acetyltransferase (pta) expression. In conclusion, strain KW023 efficiently produced 39.7 g/L of l-trp with a conversion rate of 16.7% and a productivity of 1.6 g/L/h in a 5 L fed-batch fermentation system.     

Rapid and stable production of 2,3-butanediol by an engineered <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> strain in a continuous airlift bioreactor

Utilization of renewable feedstocks for the production of bio-based bulk chemicals, such as 2,3-butanediol (2,3-BDO), by engineered strains of the non-pathogenic yeast, Saccharomyces cerevisiae, has recently become an attractive option. In this study, to realize rapid production of 2,3-BDO, a flocculent, 2,3-BDO-producing S. cerevisiae strain YPH499/dPdAdG/BDN6-10/FLO1 was constructed from a previously developed 2,3-BDO-producing strain. Continuous 2,3-BDO fermentation was carried out by the flocculent strain in an airlift bioreactor. The strain consumed more than 90 g/L of glucose, which corresponded to 90% of the input, and stably produced more than 30 g/L of 2,3-BDO over 380 h. The maximum 2,3-BDO productivity was 7.64 g/L/h at a dilution rate of 0.200/h, which was higher than the values achieved by continuous fermentation using pathogenic bacteria in the previous reports. These results demonstrate that continuous 2,3-BDO fermentation with flocculent 2,3-BDO-producing S. cerevisiae is a promising strategy for practical 2,3-BDO production.     

<Emphasis Type="Italic">Lactobacillus futsaii</Emphasis> CS3, a New GABA-Producing Strain Isolated from Thai Fermented Shrimp (<Emphasis Type="Italic">Kung</Emphasis>-<Emphasis Type="Italic">Som</Emphasis>)

Kung-Som is a popular traditional Thai fermented shrimp product. It is rich in glutamic acid, which is the major substrate for the biosynthesis of gamma-aminobutyric acid (GABA) by lactic acid bacteria (LAB). In the present study, LAB from Kung-Som were isolated, screened for GABA formation, and the two isolates that transform glutamic acid most efficiently into GABA were identified. Based on the API-CHL50 fermentation profile and a phylogenetic tree of 16S rDNA sequences, strain CS3 and CS5 were identified as Lactobacillus futsaii, which was for the first time shown to be a promising GABA producer. L. futsaii CS3 was the most efficient microorganism for the conversion of 25 mg/mL monosodium glutamate (MSG) to GABA, with a maximum yield of more than 99% conversion rate within 72 h. The open reading frame (ORF) of the glutamate decarboxylase (gad) gene was identified by PCR. It consists of 1410 bp encoding a polypeptide of 469 amino acids with a predicted molecular weight of 53.64 kDa and an isoelectric point (pI) of 5.56. Moreover, a good quality of the constructed model of L. futsaii CS3 was also estimated. Our results indicate that L. futsaii CS3 could be of interest for the production of GABA-enriched foods by fermentation and for other value-added products.     

Metabolic engineering for high glycerol production by the anaerobic cultures of <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

Glycerol is used by the cosmetic, paint, automotive, food, and pharmaceutical industries and for production of explosives. Currently, glycerol is available in commercial quantities as a by-product from biodiesel production, but the purity and the cost of its purification are prohibitive. The industrial production of glycerol by glucose aerobic fermentation using osmotolerant strains of the yeasts Candida sp. and Saccharomyces cerevisiae has been described. A major drawback of the aerobic process is the high cost of production. For this reason, the development of yeast strains that effectively convert glucose to glycerol anaerobically is of great importance. Due to its ability to grow under anaerobic conditions, the yeast S. cerevisiae is an ideal system for the development of this new biotechnological platform. To increase glycerol production and accumulation from glucose, we lowered the expression of TPI1 gene coding for triose phosphate isomerase; overexpressed the fused gene consisting the GPD1 and GPP2 parts coding for glycerol-3-phosphate dehydrogenase and glycerol-3-phosphate phosphatase, respectively; overexpressed the engineered FPS1 gene that codes for aquaglyceroporin; and overexpressed the truncated gene ILV2 that codes for acetolactate synthase. The best constructed strain produced more than 20 g of glycerol/L from glucose under micro-aerobic conditions and 16 g of glycerol/L under anaerobic conditions. The increase in glycerol production led to a drop in ethanol and biomass accumulation.     

Ex situ product recovery for enhanced butanol production by <Emphasis Type="Italic">Clostridium beijerinckii</Emphasis>

In situ butanol recovery fermentation has been intensively studied as an effective alternative to conventional butanol production, which is limited due to the cellular toxicity of butanol. However, the low biocompatibility of adsorbents often leads to failure of in situ recovery fermentations. In this study, Clostridium beijerinckii NCIMB 8052 was cultured in flasks without shaking and in situ recovery fermentation was performed by using an adsorbent L493. The amounts of acetone, butanol, and ethanol (ABE) increased by 34.4 % in the presence of the adsorbent. In contrast, cell growth and production of organic acids and ABE were retarded in the 7-L batch fermentations with in situ butanol recovery. Cell damage occurred in the fermentor upon agitation in the presence of the adsorbent, unlike in static flask cultures with in situ recovery. Ex situ recovery fermentation using circulation of fermentation broth after mid-exponential phase of cell growth was developed to avoid adsorbent-cell incompatibility. No apparent cell damage was observed and 25.7 g/L of ABE was produced from 86.2 g/L glucose in the fed-batch mode using 7 L fermentors. Thus, ex situ recovery fermentation with C. beijerinckii is effective for enhancing butanol fermentation.