Construction of a versatile in vitro cultivation screening platform using human oral microbiota.

We developed a simulation model of human oral microbiota using Bio Palette oral medium (BPOM) containing 0.02% glucose and lower bacterial nitrogen sources, derived from saliva and dental plaque. By decreasing the concentration of Gifu anaerobic medium (GAM) from 30 to 10 g L-1 , we observed increased ratios of target pathogenic genera, Porphyromonas and Fusobacterium from 0.5% and 1.7% to 1.2% and 3.5%, respectively, in the biofilm on hydroxyapatite (HA) discs. BPOM exhibited the higher ratios of Porphyromonas and Fusobacterium, and amplicon sequence variant number on HA, compared with GAM, modified GAM and basal medium mucin. Mixing glycerol stocks of BPOM culture solutions from four human subjects resulted in comparable ratios of these bacteria to the original saliva. In this simulation model, sitafloxacin showed higher inhibitory effects on P. gingivalis than minocycline hydrochloride at a low dosage of 0.1 µg mL-1 . Probiotics such as Streptococcus salivarius and Limosilactobacillus fermentum also showed significant decreases in Porphyromonas and Fusobacterium ratios on HA, respectively. Overall, the study suggests that BPOM with low carbon and nutrients could be a versatile platform for assessing the efficacy of antibiotics and live biotherapeutics in treating oral diseases caused by Porphyromonas and Fusobacterium.

Biotransformation of ferulic acid to protocatechuic acid by Corynebacterium glutamicum ATCC 21420 engineered to express vanillate O-demethylase.

Ferulic acid (4-hydroxy-3-methoxycinnamic acid, FA) is a lignin-derived phenolic compound abundant in plant biomass. The utilization of FA and its conversion to valuable compounds is desired. Protocatechuic acid (3,4-dihydroxybenzoic acid, PCA) is a precursor of polymers and plastics and a constituent of food. A microbial conversion system to produce PCA from FA was developed in this study using a PCA-producing strain of Corynebacterium glutamicum F (ATCC 21420). C. glutamicum strain F grown at 30 °C for 48 h utilized 2 mM each of FA and vanillic acid (4-hydroxy-3-methoxybenzoic acid, VA) to produce PCA, which was secreted into the medium. FA may be catabolized by C. glutamicum through proposed (I) non-ß-oxidative, CoA-dependent or (II) ß-oxidative, CoA-dependent phenylpropanoid pathways. The conversion of VA to PCA is the last step in each pathway. Therefore, the vanillate O-demethylase gene (vanAB) from Corynebacterium efficiens NBRC 100395 was expressed in C. glutamicum F (designated strain FVan) cultured at 30 °C in AF medium containing FA. Strain C. glutamicum FVan converted 4.57 ± 0.07 mM of FA into 2.87 ± 0.01 mM PCA after 48 h with yields of 62.8% (mol/mol), and 6.91 mM (1064 mg/L) of PCA was produced from 16.0 mM of FA after 12 h of fed-batch biotransformation. Genomic analysis of C. glutamicum ATCC 21420 revealed that the PCA-utilization genes (pca cluster) were conserved in strain ATCC 21420 and that mutations were present in the PCA importer gene pcaK.

Production of protocatechuic acid by Corynebacterium glutamicum expressing chorismate-pyruvate lyase from Escherichia coli.

Protocatechuic acid (3,4-dihydroxybenzoic acid; PCA) serves as a building block for polymers and pharmaceuticals. In this study, the biosynthetic pathway for PCA from glucose was engineered in Corynebacterium glutamicum. The pathway to PCA-employed elements of the chorismate pathway by using chorismate-pyruvate lyase (CPL) and 4-hydroxybenzoate hydroxylase (4-HBA hydroxylase). As C. glutamicum has the potential to synthesize the aromatic amino acid intermediate chorismate and possesses 4-HBA hydroxylase, we focused on expressing Escherichia coli CPL in a phenylalanine-producing strain of C. glutamicum ATCC21420. To secrete PCA, the gene (ubiC) encoding CPL from E. coli was expressed in C. glutamicum ATCC 21420 (strain F(UbiC)). The formation of 28.8 mg/L of extracellular 4-HBA (36 h) and 213 ± 29 mg/L of extracellular PCA (80 h) was obtained by the C. glutamicum strain F(UbiC) from glucose. The strain ATCC21420 was also found to produce extracellular PCA. PCA fermentation was performed using C. glutamicum strain F(UbiC) in a bioreactor at the optimized pH of 7.5. C. glutamicum F(UbiC) produced 615 ± 2.1 mg/L of PCA from 50 g/L of glucose after 72 h. Further, fed-batch fermentation of PCA by C. glutamicum F(UbiC) was performed with feedings of glucose every 24 h. The maximum production of PCA (1140.0 ± 11.6 mg/L) was achieved when 117.0 g/L of glucose was added over 96 h of fed-batch fermentation.

p-Hydroxycinnamic acid production directly from cellulose using endoglucanase- and tyrosine ammonia lyase-expressing Streptomyces lividans.

BACKGROUND: p-Hydroxycinnamic acid (pHCA) is an aromatic compound that serves as a starting material for the production of many commercially valuable chemicals, such as fragrances and pharmaceuticals, and is also used in the synthesis of thermostable polymers. However, chemical synthesis of pHCA is both costly and harmful to the environment. Although pHCA production using microbes has been widely studied, there remains a need for more cost-effective methods, such as the use of biomass as a carbon source. In this study, we produced pHCA using tyrosine ammonia lyase-expressing Streptomyces lividans. In order to improve pHCA productivity from cellulose, we constructed a tyrosine ammonia lyase- and endoglucanase (EG)-expressing S. lividans transformant and used it to produce pHCA from cellulose. RESULTS: A Streptomyces lividans transformant was constructed to express tyrosine ammonia lyase derived from Rhodobacter sphaeroides (RsTAL). The transformant produced 786 or 736 mg/L of pHCA after 7 days of cultivation in medium containing 1% glucose or cellobiose as the carbon source, respectively. To enhance pHCA production from phosphoric acid swollen cellulose (PASC), we introduced the gene encoding EG into RsTAL-expressing S. lividans. After 7 days of cultivation, this transformant produced 753, 743, or 500 mg/L of pHCA from 1% glucose, cellobiose, or PASC, respectively. CONCLUSIONS: RsTAL-expressing S. lividans can produce pHCA from glucose and cellobiose. Similarly, RsTAL- and EG-expressing S. lividans can produce pHCA from glucose and cellobiose with excess EG activity remaining in the supernatant. This transformant demonstrated improved pHCA production from cellulose. Further enhancements in the cellulose degradation capability of the transformant will be necessary in order to achieve further improvements in pHCA production from cellulose.

Establishment of a pure culture of the hitherto uncultured unicellular cyanobacterium Aphanothece sacrum, and phylogenetic position of the organism.

Aphanothece sacrum, an edible freshwater unicellular cyanobacterium, was isolated by using novel synthetic media (designated AST and AST-5xNP). The media were designed on the basis of the ratio of inorganic elements contained in A. sacrum cells cultured in a natural pond. The isolated strain exhibits unicellular rod-shaped cells approximately 6 microm in length that are scattered in an exopolysaccharide matrix, a feature similar to that of natural A. sacrum. DNA analysis of the isolated strain revealed that it carried two ferredoxin genes whose deduced amino acid sequences were almost identical to previously published sequences of ferredoxins from natural A. sacrum. Analysis of the 16S rRNA gene and ferredoxin genes revealed that A. sacrum occupies a phylogenetically unique position among the cyanobacteria.