One-Pot Synthesis of Phenylglyoxylic Acid from Racemic Mandelic Acids via Cascade Biocatalysis.

Phenylglyoxylic acid (PGA) are key building blocks and widely used to synthesize pharmaceutical intermediates or food additives. However, the existing synthetic methods for PGA generally involve toxic cyanide and complex processes. To explore an alternative method for PGA biosynthesis, we envisaged cascade biocatalysis for the one-pot synthesis of PGA from racemic mandelic acid. A novel mandelate racemase named ArMR showing higher expression level (216.9 U·mL-1 fermentation liquor) was cloned from Agrobacterium radiobacter and identified, and six recombinant Escherichia coli strains were engineered to coexpress three enzymes of mandelate racemase, d-mandelate dehydrogenase and l-lactate dehydrogenase, and transform racemic mandelic acid to PGA. Among them, the recombinant E. coli TCD 04, engineered to coexpress three enzymes of ArMR, LhDMDH, and LhLDH, can transform racemic mandelic acid (100 mM) to PGA with 98% conversion. Taken together, we provide a green approach for one-pot biosynthesis of PGA from racemic mandelic acid.

Biosynthesis of Phenylglyoxylic Acid by LhDMDH, a Novel d-Mandelate Dehydrogenase with High Catalytic Activity.

d-Mandelate dehydrogenase (DMDH) has the potential to convert d-mandelic acid to phenylglyoxylic acid (PGA), which is a key building block in the field of chemical synthesis and is widely used to synthesize pharmaceutical intermediates or food additives. A novel NAD+-dependent d-mandelate dehydrogenase was cloned from Lactobacillus harbinensi (LhDMDH) by genome mining and expressed in Escherichia coli BL21. After being purified to homogeneity, the oxidation activity of LhDMDH toward d-mandelic acid was approximately 1200 U·mg-1, which was close to four times the activity of the probe. Meanwhile, the kcat/ Km value of LhDMDH was 28.80 S-1·mM-1, which was distinctly higher than the probe. By coculturing two E. coli strains expressing LhDMDH and LcLDH, we developed a system for the efficient synthesis of PGA, achieving a 60% theoretical yield and 99% purity without adding coenzyme or cosubstrate. Our data supports the implementation of a promising strategy for the chiral resolution of racemic mandelic acid and the biosynthesis of PGA.

The Epidemiological Characteristics of Beijing Lineage Mycobacterium tuberculosis from a National Referral Center in China.

Our study was to investigate the epidemiological characteristics of M.tuberculosis from a national tuberculosis referral center in China. All strains isolated from TB patients, were genotyped by the RD105 deletion, 8 and 51 SNP loci and VNTR. The high differentiation SNPs of modern Beijing strains were analyzed for protein function and structure. 413 M. tuberculosis were included. Of 379 Beijing lineage M. tuberculosis, 'modern' and 'ancient' strains respectively represented 85.5% (324/379) and 14.5% (55/379). Rv2494 (V48A) and Rv0245 (S103F) were confirmed as high differentiation SNPs associated with modern strains. In a word, Modern Beijing lineage M.tuberculosis was dominant and the structural models suggested that modern sub-lineage may more easily survive in 'extreme' host condition.

Differences in the population of genetics of Mycobacterium tuberculosis between urban migrants and local residents in Beijing, China.

BACKGROUND: Currently, migration has become one of the risk factors of high burden of tuberculosis in China. This study was to explore the influence of mass migration on the dynamics of Mycobacterium (M.) tuberculosis in Beijing, the capital and an urban area of China. METHODS: Three hundred and thirty-six M. tuberculosis strains from the Changping district, where the problem of urban migrants was more pronounced than in other Beijing regions, were genotyped by Spoligotyping, large sequence polymorphisms (LSPs 105 and 181), and variable number tandem repeat (VNTR) typing. Based on the genotype data, the phylogeny of the isolates was studied. RESULTS: In Changping district, the proportion of Beijing lineage M. tuberculosis isolates amounted to 89.0% (299/336), among which 86.6 % (252) belonged to the modern lineage. The frequency of modern Beijing lineage strains is so high (around 75% (252/336)) that associated risk factors affecting the tuberculosis epidemic cannot be determined. The time to the most recent common ancestor (TMRCA) of the Beijing lineage strains was estimated to be 5073 (95% CI: 4000-6200) years. There was no significant difference in the genetic variation of Beijing isolates from urban migrants and local residents. CONCLUSIONS: The clone of modern Beijing lineage M. tuberculosis, which is dominant in the Beijing area, most likely started to expand with the five thousand-year-old Chinese civilization. In the future, with the urbanization in the whole of China, modern Beijing lineage M. tuberculosis may gain the larger geographical spread.

[Origin, phylogeny, and spread of Mycobacterium tuberculosis Beijing lineage in the five provinces of northern China].

OBJECTIVE: Using methodology of molecular genetics to explore the origin, phylogen, and gene flow of Mycobacterium tuberculosis (MTB) Beijing lineage in the five provinces from northern China, including Heilongjiang, Jilin, Liaoning, Neimenggu and Ningxia. METHODS: 234 MTB Beijing lineage strains were genotyped by 24 Variable Number Tandem Repeat (VNTR), and the h (the allelic diversity) value of each VNTR locus was calculated. On individual level of phylogeny, it was constructed Neighbor-Joining (N-J) tree and minimum spanning tree (MST). Phylogenetic tree was built at the population level, and the most recent common ancestor (TMRCA) was estimated through Bayesian model. Molecular variance (AMOVA) was used to understand the gene flow among strains discovered from the five provinces. RESULTS: Allelic diversities of the 24 VNTR loci were low (h: 0.000 - 0.744). 234 strains of MTB Beijing lineage were dispersed in individual branch of the N-J tree, with 62.0% (145) of them grouped to the same "colonial complexes" in MST. At the population level, the evolution relationship of 234 strains appeared the closest to Beijing lineage, which was from MIRU-VNTRplus database, and the bootstrap was 100. The TMRCA was 5308 (95%CI: 4263 - 6470) years. Differences of pairwise Fst values acquired by AMOVA between Jilin and Heilongjiang, Liaoning, Neimenggu and Ningxia, were not statistically significant (P > 0.05). CONCLUSION: The genetic similarity of Beijing lineage MTB from the five provinces of northern China was high. The phylogeny branches had no characteristic dispersal in each province. It was speculated that these strains showed an evolution from a clone of MTB Beijing lineage (about 5000 years ago). The gene flow was taking place between neighboring zones.