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Fig. 2 | Microbial Cell Factories

Fig. 2

From: Microbial production of vitamin B12: a review and future perspectives

Fig. 2

Design of a heterologous biosynthetic pathway. a A host for the heterologous biosynthetic pathway is selected considering the capability of precursor and cofactor supply, genetic engineering tools, and industrial-scale fermentation capability, utilizing cheap and readily available carbon sources. b Enzyme activity is verified in vitro and subsequently in vivo. Products of the in vitro assay or intracellular reaction products are detected via spectroscopic analysis, mass spectrometry, or microbiological assays. c Heterogeneous genes and other functional elements are assembled on plasmids via gene assembly methods such as SLIC, CPEC, Gibson, golden gate, DNA assembler and LCR, or integrated into the genome. To decrease the difficulty of building the metabolic pathway, it is divided into separate modules. These modules are verified sequentially in a heterologous host and then assembled. d Based on the quantification of metabolites, bottlenecks should be removed and metabolic flux should be integrated to target compound maximization. To optimize gene expression in the metabolic pathway, promoters, RBS, and gene copy number are designed and implemented at the transcriptional or translational levels. e The characteristics of the engineered strains are verified via fermentation. Various substrates (e.g., ALA, cobalt ions, betaine and DMB) and varying conditions (e.g., dissolved oxygen concentration, pH, and temperature) can be optimized to improve yield and productivity

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