Engineering CrtW and CrtZ for improving biosynthesis of astaxanthin in Escherichia coli.

This study engineered ß-carotene ketolase CrtW and ß-carotene hydroxylase CrtZ to improve biosynthesis of astaxanthin in Escherichia coli. Firstly, crtW was randomly mutated to increase CrtW activities on conversion from ß-carotene to astaxanthin. A crtW* mutant with A6T, T105A and L239M mutations has improved 5.35-fold astaxanthin production compared with the wild-type control. Secondly, the expression levels of crtW* and crtZ on chromosomal were balanced by simultaneous modulation RBS regions of their genes using RBS library. The strain RBS54 selected from RBS library, directed the pathway exclusively towards the desired product astaxanthin as predominant carotenoid (99%). Lastly, the number of chromosomal copies of the balanced crtW-crtZ cassette from RBS54 was increased using a Cre-loxP based technique, and a strain with 30 copies of the crtW*-crtZ cassette was selected. This final strain DL-A008 had a 9.8-fold increase of astaxanthin production compared with the wild-type control. Fed-batch fermentation showed that DL-A008 produced astaxanthin as predominant carotenoid (99%) with a specific titer of 0.88 g·L-1 without addition of inducer. In conclusion, through constructing crtW mutation, balancing the expression levels between crtW* and crtZ, and increasing the copy number of the balanced crtW*-crtZ cassette, the activities of ß-carotene ketolase and ß-carotene hydroxylase were improved for conversion of ß-carotene to astaxanthin with higher efficiency. The series of conventional and novel metabolic engineering strategies were designed and applied to construct the astaxanthin hetero-producer strain of E. coli, possibly offering a general approach for the construction of stable hetero-producer strains for other natural products.

Diversity of Salmonella isolates using serotyping and multilocus sequence typing.

One hundred and twenty-one Salmonella isolates were obtained from food, feed, and live chicken samples derived from 13 countries or regions. In this study, their subtypes were evaluated by serotyping and multilocus sequence typing (MLST), and their genetic profiles were also characterized. It was demonstrated by serotyping on these isolates that 36 various serovars were obtained in this study, of which three serotypes S. Babelsberg, S. Fresno, and S. II were first found in mainland China. Based on Simpson's index of diversity, the serotyping method had a 0.943 discriminatory power. Meanwhile, there were a total of 42 unique sequence types (STs) characterized by MLST, and the discriminatory power of MLST (D = 0.947) was close to that of the serotyping method. In MLST, hisD revealed the highest levels of nucleotide diversity. In addition, ST-92 was the most common ST represented by 16 Salmonella isolates, followed by ST-367 which was represented by 14 isolates. Seven new alleles were identified, which were associated with other alleles and resulted in the assignment of nine new STs. It was concluded from the results that MLST was generally associated with serotype, but not associated with the epidemiological source of the samples, and antimicrobial resistance patterns.

Development of a quantitative polymerase chain reaction method using a live bacterium as internal control for the detection of Listeria monocytogenes.

A novel real-time quantitative polymerase chain reaction (Q-PCR) assay was developed for rapid and accurate detection of Listeria monocytogenes. In this Q-PCR assay, a computational DNA random shuffling method was used to design an internal amplification control (IAC) sequence, which was the same in length and G + C content to the hly amplicon. This IAC sequence was inserted into the genome of L. monocytogenes to create a mutant strain named L. monocytogenes-IAC. The LM-IAC was used as an internal control during the PCR assay and produced accurate quantification of L. monocytogenes due to similar DNA extraction and amplification efficiencies between LM-IAC strain and wild-type L. monocytogenes. Quantification by this method was over a 5-log linearity range of initial L. monocytogenes with an R(2) value of 0.9997. This PCR method will provide accurate quantification of L. monocytogenes and can be used in the clinic and food assays for diagnostic purposes.