Global warming and depletion of fossil fuels are driving research on the production of compounds and biofuels using microbial cell factories rather than chemical synthesis. In previous studies, to obtain high-efficiency foreign genes from genetic information they were searched in a database, obtained by degenerating PCR using degenerate primer or by using a fosmid library construction. However, these old methods are costly and often inaccurate. Recent development of next generation sequencing can be used to quickly and easily obtain information on high-efficiency genes. Metabolic engineering is implemented for developing microbial cell factories using genetic information from next generation sequencing. Construction of heterologous biosynthesis pathways is possible by using genetic information. Many microorganisms such as E. coli or yeast can be used to produce valuable materials on an industrial scale in an efficient manner.
A noble type of strain, Planococcus faecalis AJ003T, isolated from the feces of the Antarctic penguin synthesizes a rare C30 carotenoid, glycosyl-4,4'-diapoinurosporphorin-4'-ol-4-ol acid. The genome of P. faecalis AJ003T comprises a 3,495,892bp circular chromosome (40.9 % G+C content), and is devoid of any extrachromosomal plasmids. Genome annotation analysis revealed that it six genes involved in the carotenoid pathway. The function and complementation of 4,4'-diapophytoene synthase (CrtM), 4,4'-diapophytoene desaturase (CrtN), 4,4-diaponeurosporene oxidase (CrtP) and aldehyde dehydrogenase (aldH) were analyzed in E. coli. Complementation of each gene of P. faecalis AJ003T with carotenoid biosynthetic module of Staphylococcus aureus in E. coli assigned the function of each gene. As a result, 4,4′-diaponeurosporenoic acid, 4,4′-diapolycopene-4,4’-dioic acid, 4,4′-diapolycopen-4′-al-4-oic acid and 4,4′-diapolycopenoic acid were detected by HPLC analysis. In conclusion, by using NGS, the C30 carotenoid gene was efficiently obtained and microbial cell factories were constructed by the metabolic engineering technique to produce 4,4'-diapolycopene-4,4'-dioic acid and 4,4'-diaponeurosporenoic acid metabolic pathway.
A novel species, Flavobacterium kingsejong WV39T, isolated from the feces of the Antarctic penguin overproduces zeaxanthin as a main carotenoid. The complete genome of F. kingsejong WV39T is made up of a single circular chromosome (4,224,053 bp, 39.8% G +C content). Genome annotation analysis revealed that it has a five zeaxanthin genes and six mevalonate pathway genes that produce the IPP, a precursor of zeaxanthin. Functional analysis of the mevalonate enzyme from F. kingsejong WV39T in E. coli was completed. Each gene of F. kingsejong WV39T was complemented with the mevalonate biosynthesis module of Enterococcus faecalis and Flavobacterium faecalis WV33 in E. coli. As a result, the titer, yield and productivity of mevalonate were 64.02 ± 1.43 g/L, 0.24 ± 0 g/g and 1.06 ± 0 g/L/h, respectively, through fermentation using the plasmid system and pH stat method. To develop industrial strains that overcome the instability of plasmids, the 5' untranslated region (5' UTR) of mRNA was introduced for the genome editing, mRNA expression and stability of E coli. The mevalonate titer, yield and productivity of the strain was 14.07 ± 0.12 g/L, 0.21 ± 0.01 g/g and 0.46 ± 0.01 g/L/ h, respectively. The yield increased by about 7 folds as compared with that of the strain editing of mevalonate gene. In conclusion, the mevalonate pathway gene found in NGS was introduced into E. coli and the possibility of producing mevalonate was showed by flask culture and fed batch.
In conclusion, we succeeded in securing high efficiency genes easily and quickly by using the next generation sequencing and confirmed the function of genes by introducing them into E. coli. These results using engineering tools will provide information for further application and engineering of carotenoids and mevalonate.