Sequence analysis of <Emphasis Type="Italic">KmXYL1</Emphasis> genes and verification of thermotolerant enzymatic activities of xylose reductase from four <Emphasis Type="Italic">Kluyveromyces marxianus</Emphasis> strains

Kluyveromyces marxianus has the capability of producing xylitol from xylose because of the endogenous xylose reductase (KmXYL1) gene. In this study, we cloned KmXYL1 genes and compared amino acid sequences of xylose reductase (XR) from four K. marxianus strains (KCTC 7001, KCTC 7155, KCTC 17212, and KCTC 17555). Four K. marxianus strains showed high homologies (99%) of amino acid sequences with those from other reported K. marxianus strains and around 60% homologies with that from Scheffersomyces stipitis. For XR enzymatic activities, four K. marxianus strains exhibited thermostable XR activities up to 45°C and K. marxianus KCTC 7001 showed the highest XR activity. When reaction temperatures were increased from 30 to 45°C, NADH-dependent XR activity from K. marxianus KCTC 7001 was highly increased (46%). When xylitol fermentations were performed at 30 or 45°C, four K. marxianus strains showed very poor xylitol production capabilities regardless fermentation temperatures. Xylitol productions from four K. marxianus strains might be limited because of low xylose uptake rate or cell growth although they have high thermostable XR activities.     

Polymorphism of lactose genes in the dairy yeasts <Emphasis Type="Italic">Kluyveromyces marxianus</Emphasis>, potential probiotic microorganisms

Molecular karyotyping and Southern blot hybridization were used to investigate chromosomal polymorphism of the LAC genes controlling lactose fermentation in Kluyveromyces marxianus strains isolated from various dairy products and natural sources in Russia and CIS countries. Profound polymorphism of karyotype patterns and accumulation of LAC genes were observed in dairy K. marxianus strains. K. marxianus strains isolated from dairy products intensively fermented lactose at 37°C after one day of cultivation, while non-dairy strains exhibited delayed lactose fermentation or did not ferment it at all. Based on the fermentation tests, twelve K. marxianus strains were selected, which are of interest as potential probiotic microorganisms suitable for further molecular genetic studies and breeding.     

<Superscript>13</Superscript>C metabolite profiling to compare the central metabolic flux in two yeast strains

¹³C metabolite profiling to quantify the dynamic changes of central carbon metabolites was attempted using mass isotopomer distribution analysis in two yeast strains, Saccharomyces cerevisiae and Kluyveromyces marxianus. Mass and isotopomer balances of the intermediates were examined and calculated in both yeast species and central carbon metabolic fluxes were successfully determined. Metabolic fluxes of pentose phosphate pathway in K. marxianus were 1.66 times higher than S. cerevisiae. The flux difference was also supported by relatively high abundance of partially labeled fructose 6-phosphate and 3-phosphoglycerate as well as an increased concentration of labeled L-valine in K. marxianus. Metabolic flux analysis combined with dynamic metabolite profiling has provided better understanding in the central carbon metabolic pathways of two model organisms and can be applied as a method to analyze more complicated metabolic networks in other organisms.     

Modeling and parameters identification of 2-keto-<Emphasis Type="SmallCaps">l</Emphasis>-gulonic acid fed-batch fermentation

This article presents a modeling approach for industrial 2-keto-l-gulonic acid (2-KGA) fed-batch fermentation by the mixed culture of Ketogulonicigenium vulgare (K. vulgare) and Bacillus megaterium (B. megaterium). A macrokinetic model of K. vulgare is constructed based on the simplified metabolic pathways. The reaction rates obtained from the macrokinetic model are then coupled into a bioreactor model such that the relationship between substrate feeding rates and the main state variables, e.g., the concentrations of the biomass, substrate and product, is constructed. A differential evolution algorithm using the Lozi map as the random number generator is utilized to perform the model parameters identification, with the industrial data of 2-KGA fed-batch fermentation. Validation results demonstrate that the model simulations of substrate and product concentrations are well in coincidence with the measurements. Furthermore, the model simulations of biomass concentrations reflect principally the growth kinetics of the two microbes in the mixed culture.     

Regulation of <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> genetic engineering on the production of acetate esters and higher alcohols during Chinese Baijiu fermentation

Acetate esters and higher alcohols greatly influence the quality and flavor profiles of Chinese Baijiu (Chinese liquor). Various mutants have been constructed to investigate the interactions of ATF1 overexpression, IAH1 deletion, and BAT2 deletion on the production of acetate esters and higher alcohols. The results showed that the overexpression of ATF1 under the control of the PGK1 promoter with BAT2 and IAH1 double-gene deletion led to a higher production of acetate esters and a lower production of higher alcohols than the overexpression of ATF1 with IAH1 deletion or overexpression of ATF1 with BAT2 deletion. Moreover, deletion of IAH1 in ATF1 overexpression strains effectively increased the production of isobutyl acetate and isoamyl acetate by reducing the hydrolysis of acetate esters. The decline in the production of higher alcohol by the ATF1 overexpression strains with BAT2 deletion is due to the interaction of ATF1 overexpression and BAT2 deletion. Mutants with varying abilities of producing acetate esters and higher alcohols were developed by genetic engineering. These strains have great potential for industrial application.     

Two propanediol utilization-like proteins of <Emphasis Type="Italic">Moorella thermoacetica</Emphasis> with phosphotransacetylase activity

Moorella thermoacetica is one of the model acetogenic bacteria for the resolution of the Wood–Ljungdahl (acetyl-CoA) pathway in which CO₂ is autotrophically assimilated yielding acetyl-CoA as central intermediate. Its further conversion into acetate relies on subsequent phosphotransacetylase (PTA) and acetate kinase reactions. However, the genome of M. thermoacetica contains no pta homologous gene. It has been speculated that the moth_0864 and moth_1181 gene products sharing similarities with an evolutionarily distinct phosphotransacylase involved in 1,2-propanediol utilization (PDUL) of Salmonella enterica act as PTAs in M. thermoacetica. Here, we demonstrate specific PTA activities with acetyl-CoA as substrate of 9.05 and 2.03 U/mg for the recombinant enzymes PDUL1 (Moth_1181) and PDUL2 (Moth_0864), respectively. Both showed maximal activity at 65 °C and pH 7.6. Native proteins (90 kDa) are homotetramers composed of four subunits with apparent molecular masses of about 23 kDa. Thus, one or both PDULs of M. thermoacetica might act as PTAs in vivo catalyzing the penultimate step of the Wood–Ljungdahl pathway toward the formation of acetate. In silico analysis underlined that up to now beside of M. thermoacetica, only Sporomusa ovata contains only PDUL like class^III-PTAs but no other phosphotransacetylases or phosphotransbutyrylases (PTBs).     

Immobilized <Emphasis Type="Italic">Kluyveromyces marxianus</Emphasis> cells in carboxymethyl cellulose for production of ethanol from cheese whey: experimental and kinetic studies

Cheese whey fermentation to ethanol using immobilized Kluyveromyces marxianus cells was investigated in batch and continuous operation. In batch fermentation, the yeast cells were immobilized in carboxymethyl cellulose (CMC) polymer and also synthesized graft copolymer of CMC with N-vinyl-2-pyrrolidone, denoted as CMC-g-PVP, and the efficiency of the two developed cell entrapped beads for lactose fermentation to ethanol was examined. The yeast cells immobilized in CMC-g-PVP performed slightly better than CMC with ethanol production yields of 0.52 and 0.49 g ethanol/g lactose, respectively. The effect of supplementation of cheese whey with lactose (42, 70, 100 and 150 g/l) on fermentative performance of K. marxianus immobilized in CMC beads was considered and the results were used for kinetic studies. The first order reaction model was suitable to describe the kinetics of substrate utilization and modified Gompertz model was quite successful to predict the ethanol production. For continuous ethanol fermentation, a packed-bed immobilized cell reactor (ICR) was operated at several hydraulic retention times; HRTs of 11, 15 and 30 h. At the HRT of 30 h, the ethanol production yield using CMC beads was 0.49 g/g which implies that 91.07 % of the theoretical yield was achieved.     

Cyclic utilization of HP-β-CD in the bioconversion of cortisone acetate by <Emphasis Type="Italic">Arthrobacter simplex</Emphasis>

Objective

To establish a method for the recovery and reutilization of hydroxypropyl-β-cyclodextrin (HP-β-CD) to lower the cost of its industrial application in cortisone acetate bioconversion.

Results

HP-β-CD is not degraded by Arthrobacter simplex CPCC140451 (ASP) resting cells and 96.4 % HP-β-CD could be recovered by isobutyl acetate extraction. Moreover, the inclusion ability of recovered HP-β-CD barely decreased. The saccharide metabolic and catalytic activities of ASP were greater in the aqueous phase after extracting with isobutyl acetate than other organic solvents. Cyclic utilization tests showed that cortisone acetate conversion ratio was 91.0 % after eight cycles and reached 95.7 % with 0.2–0.6 mM HP-β-CD. Furthermore, >90 % conversion ratio was reached per cycle through a co-cyclic-utilization method with HP-β-CD and immobilized ASP.

Conclusion

Cortisone acetate conversion ratio in the HP-β-CD cyclic-utilization method is promising for industrial applications. The method can also be expanded to other CDs and other hydrophobic compounds bioconversion.

     

Investigation of microorganisms involved in kefir biofilm formation

Kefir is a natural fermentation agent composed of various microorganisms. To address the mechanism of kefir grain formation, we investigated the microbial role in forming kefir biofilms. The results showed that a biofilm could be formed in kefir-fermented milk and the biofilm forming ability reached the maximum at 13 days. The strains Kluyveromyces marxianus, Lactococcus lactis, Leuconostoc mesenteroides, Lactobacillus kefiri, Lactobacillus sunkii and Acetobacter orientalis were isolated from kefir biofilms by the streak-plate method. These microorganisms were analysed with respect to biofilm forming properties, including their surface characterisation (hydrophobicity and zeta potentials) and the microbial aggregation. The results indicated that Klu. marxianus possessed the strongest biofilm forming properties with the strongest hydrophobicity, lowest zeta potential and greatest auto-aggregation ability. When Klu. marxianus and Ac. orientalis were co-cultured with kefir LAB strains respectively, it was found that mixing Klu. marxianus with Lb. sunkii produced the highest co-aggregation ability. These results elucidated the mechanism of kefir biofilm formation and the microorganisms involved.     

<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 %.     

Microbial production of ethanol from acetate by engineered <Emphasis Type="Italic">Ralstonia eutropha</Emphasis>

This study was performed to produce ethanol from acetate using a genetically engineered Ralstonia eutropha. In order to genetically modify R. eutropha H16, phaCAB operon encoding metabolic pathway genes from acetyl-CoA to polyhydroxybutyrate (PHB) was deleted and adhE encoding an alcohol dehydrogenase from Escherichia coli was overexpressed for conversion of acetyl-CoA to ethanol. The resulting strain produced ethanol up to 170 mg/L when cultivated in minimal media supplemented with 5 g/L of acetate as a sole carbon source. Growth and ethanol production were optimized by adjusting nitrogen source (NH₄Cl) content and repetitive feeding of acetate into the bacterial culture, by which the ethanol production was reached to approximately 350 mg/L for 84 h.     

Development of schemes of induced mutagenesis for improving the productivity of <Emphasis Type="Italic">Aspergillus</Emphasis> strains producing amylolytic enzymes

In order to improve the biosynthesis of amylolytic enzymes by industrial Aspergillus strains, the efficiency of stepwise application of the following methods of induced mutagenesis was studied: ultraviolet (UV) irradiation, gamma irradiation, and treatment with N-methyl-N-nitro-N-nitrosoguanidine (NG). It was found that at the early stages of mutagenesis of the glucoamylase-producing A. awamori strain UV and NG, used either alone or in combination, were efficient enough as mutagenic agents, providing an increase of glucoamylase activity by 30–35 and 50–60%, respectively. At the later stages, serial UV mutagenesis and gamma-irradiation showed high efficiency for both A. awamori, and A. oryzae strains. Gamma-mutagenesis of Aspergillus strains using a cobalt source provided the most stable and highly active strains retaining 90–95% of their activity after five transfers on agar medium. The experiments resulted in significant improvement of the studied industrial strains, more than doubling activity of the target enzymes.     

Growth and wax ester production of an <Emphasis Type="Italic">Acinetobacter baylyi</Emphasis> ADP1 mutant deficient in exopolysaccharide capsule synthesis

Acinetobacter baylyi ADP1 naturally produces wax esters that could be used as a raw material in industrial applications. We attempted to improve wax ester yield of A. baylyi ADP1 by removing rmlA, a gene involved in exopolysaccharide production. Growth rate, biomass formation and wax ester yield on 4-hydroxybenzoate were not affected, but the rmlA ^? strain grew slower on acetate, while reaching similar biomass and wax ester yield. The rmlA ^? cells had malformed shape and large size and grew poorly on glucose without expression of the gene for pyruvate kinase (pykF) from Escherichia coli. The pykF-expressing rmlA ^? strain had similar growth rate, lowered biomass formation and improved wax ester production on glucose as compared to the wild-type strain expressing pykF. Cultivation of the pykF-expressing rmlA ^? strain on an elevated glucose concentration in a medium supplemented with amino acids resulted in doubled molar wax ester yield and acetate production.     

Metabolism of the thermophilic bacterium <Emphasis Type="Italic">Oceanithermus profundus</Emphasis>

The metabolism of the novel facultatively anaerobic thermophilic bacterium Oceanithermus profundus was studied during growth on maltose, acetate, pyruvate, and hydrogen. The utilization of carbohydrates was shown to proceed via the glycolytic pathway. Under microaerobic growth conditions, the metabolism of O. profundus grown on maltose depended on the substrate concentration. At an initial maltose concentration of 1.4 mM, O. profundus carried out oxygen respiration, and in the presence of 3.5 mM maltose, facilitated fermentation occurred, with the formation of acetate and ethanol and limited involvement of oxygen. The use of pyruvate and acetate occurred via the TCA cycle. In cells grown on acetate, the activity of glyoxylate pathway enzymes was revealed. Depending on the energy-yielding process providing for growth (oxygen respiration or nitrate reduction), cells contained cytochromes a and c or b, respectively. The results obtained demonstrate the plasticity of the metabolism of O. profundus, which thus appears to be well-adjusted to the rapidly changing conditions in deep-sea hydrothermal vents.     

Persistence of <Emphasis Type="Italic">Methanosaeta</Emphasis> populations in anaerobic digestion during process instability

Anaerobic digestion is a sustainable technology for the treatment of organic waste and production of biogas. Acetoclastic methanogenesis accounts for the majority of methane production in anaerobic digestion. Therefore, sustaining robust acetoclastic methanogens is important for stable process performance. Due to faster growth kinetics at high acetate concentrations, it has been considered that Methanosarcina would be more prevalent than Methanosaeta in unstable anaerobic digestion processes which frequently experience high acetate levels. Methanogen population dynamics were monitored in multiple continuous anaerobic digesters for 500 days. Results from quantitative polymerase chain reaction analysis show that Methanosaeta dominated over Methanosarcina in anaerobic digestion at high acetate levels up to 44 mM, suggesting the potential of Methanosaeta as a robust and efficient acetoclastic candidate for resilient anaerobic methane conversion. Further efforts are needed to identify mechanisms contributing to the unexpected competitiveness of these methanogens at high acetate levels observed in this study.     

Contribution of glutamate decarboxylase in <Emphasis Type="Italic">Lactobacillus reuteri</Emphasis> to acid resistance and persistence in sourdough fermentation

Background

Acid stress impacts the persistence of lactobacilli in industrial sourdough fermentations, and in intestinal ecosystems. However, the contribution of glutamate to acid resistance in lactobacilli has not been demonstrated experimentally, and evidence for the contribution of acid resistance to the competitiveness of lactobacilli in sourdough is lacking. It was therefore the aim of this study to investigate the ecological role of glutamate decarboxylase in L. reuteri.

Results

A gene coding for a putative glutamate decarboxylase, gadB, was identified in the genome of L. reuteri 100-23. Different from the organization of genetic loci coding for glutamate decarboxylase in other lactic acid bacteria, gadB was located adjacent to a putative glutaminase gene, gls3. An isogenic deletion mutant, L. reuteri ?gadB, was generated by a double crossover method. L. reuteri 100-23 but not L. reuteri ?gadB converted glutamate to γ-aminobutyrate (GABA) in phosphate butter (pH 2.5). In sourdough, both strains converted glutamine to glutamate but only L. reuteri 100-23 accumulated GABA. Glutamate addition to phosphate buffer, pH 2.5, improved survival of L. reuteri 100-23 100-fold. However, survival of L. reuteri ?gadB remained essentially unchanged. The disruption of gadB did not affect growth of L. reuteri in mMRS or in sourdough. However, the wild type strain L. reuteri 100-23 displaced L. reuteri ?gadB after 5 cycles of fermentation in back-slopped sourdough fermentations.

Conclusions

The conversion of glutamate to GABA by L. reuteri 100-23 contributes to acid resistance and to competitiveness in industrial sourdough fermentations. The organization of the gene cluster for glutamate conversion, and the availability of amino acids in cereals imply that glutamine rather than glutamate functions as the substrate for GABA formation. The exceptional coupling of glutamine deamidation to glutamate decarboxylation in L. reuteri likely reflects adaptation to cereal substrates.

     

Progress in the microbial production of <Emphasis Type="Italic">S</Emphasis>-adenosyl-<Emphasis Type="SmallCaps">l</Emphasis>-methionine

S-Adenosyl-l-methionine (SAM), which exists in all living organisms, serves as an activated group donor in a range of metabolic reactions, including trans-methylation, trans-sulfuration and trans-propylamine. Compared with its chemical synthesis and enzyme catalysis production, the microbial production of SAM is feasible for industrial applications. The current clinical demand for SAM is constantly increasing. Therefore, vast interest exists in engineering the SAM metabolism in cells for increasing product titers. Here, we provided an overview of updates on SAM microbial productivity improvements with an emphasis on various strategies that have been used to enhance SAM production based on increasing the precursor and co-factor availabilities in microbes. These strategies included the sections of SAM-producing microbes and their mutant screening, optimization of the fermentation process, and the metabolic engineering. The SAM-producing strains that were used extensively were Saccharomyces cerevisiae, Pichia pastoris, Candida utilis, Scheffersomyces stipitis, Kluyveromyces lactis, Kluyveromyces marxianus, Corynebacterium glutamicum, and Escherichia coli, in addition to others. The optimization of the fermentation process mainly focused on the enhancement of the methionine, ATP, and other co-factor levels through pulsed feeding as well as the optimization of nitrogen and carbon sources. Various metabolic engineering strategies using precise control of gene expression in engineered strains were also highlighted in the present review. In addition, some prospects on SAM microbial production were discussed.     

<Emphasis Type="Italic">PiggyBac</Emphasis> transposon-mediated mutagenesis and application in yeast <Emphasis Type="Italic">Komagataella phaffii</Emphasis>

Objective

Around one-fourth of the Komagataella phaffii genes encode hypothetical proteins with unknown functions. However, lack of powerful tools for genetic screening in K. phaffii significantly limits the functional analysis of these unknown genes. Transposon mutagenesis has been utilized as an insertional mutagenesis tool in many other organisms and would be extremely valuable if it could be applied in K. phaffii.

Results

In this study, we investigated in K. phaffii the transposition activity and efficiency of piggyBac (PB) transposon, a DNA transposon from the cabbage looper moth Trichoplusia ni through the integrated-plasmid system. We also designed a binary-plasmid system which could generate stable mutants. Finally we evaluated the quality of this mutagenesis system by a simple screening for functional genes involved in K. phaffii carbon catabolite repression.

Conclusions

Our results demonstrate that PB-mediated mutagenesis could be a feasible and useful tool for functional gene screening in K. phaffii.

     

<Emphasis Type="Italic">Terminalia chebula</Emphasis> Retz. Fruit Extracts Inhibit Bacterial Triggers of Some Autoimmune Diseases and Potentiate the Activity of Tetracycline

Terminalia chebula Retz. is a northern Indian plant species known for its anti-inflammatory and antimicrobial properties. T. chebula fruit powder was extracted with solvents of varying polarity and screened for bacterial growth inhibition by disc diffusion assay. The minimum inhibitory concentration (MIC) was quantified by both liquid dilution and disc diffusion techniques. To screen for combinatorial effects, the T. chebula fruit extracts were combined with a range of conventional antibiotics and tested against each bacteria using a liquid dilution assay. Where synergy was detected, the optimal ratios were determined using isobologram analysis. Toxicity was examined using Artemia nauplii and HDF bioassays. T. chebula fruit methanolic, aqueous and ethyl acetate extracts displayed strong antimicrobial activity against the bacterial triggers of all autoimmune inflammatory diseases except K. pneumoniae, for which only moderate inhibition was observed. Indeed, MIC values as low as 195 μg/mL were measured for the aqueous extract against a resistant strain of P. aeruginosa. Of further note, both the aqueous and ethyl acetate extracts interacted synergistically in combination with tetracycline against K. pneumoniae (Σ FIC 0.38 and 0.25 respectively). All extracts were nontoxic in the Artemia and HDF toxicity assays, further indicating their potential for medicinal use.     

<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.