The Gram-positive model bacterium Bacillus subtilis , has been broadly applied in various fields because of its low pathogenicity and strong protein secretion ability, as well as its well-developed fermentation technology. B. subtilis is considered as an attractive host in the field of metabolic engineering, in particular for protein expression and secretion, so it has been well studied and applied in genetic engineering. In this review, we discussed why B. subtilis is a good chassis cell for metabolic engineering. We also summarized the latest research progress in systematic biology, synthetic biology and evolution-based engineering of B. subtilis , and showed systemic metabolic engineering expedite the harnessing B. subtilis for bioproduction.

Hydrogenobyrinic acid, a modified tetrapyrrole composed of eight five-carbon compounds, is a key intermediate and central framework of vitamin B12. Synthesis of hydrogenobyrinic acid requires eight S-adenosyl-methionine working as the methyl group donor catalyzed by 12 enzymes including six methyltransferases, causing the great shortage of S-adenosyl-methionine and accumulation of S-adenosyl-homocysteine, which is uneconomic and unsustainable for the cascade reaction. Here, we report a cell-free synthetic system for producing hydrogenobyrinic acid by integrating 12 enzymes using 5-aminolevulininate as a substrate and develop a novel S-adenosyl-methionine regeneration system to steadily supply S-adenosyl-methionine and avoid the accumulated inhibition of S-adenosyl-homocysteine by consuming a cheaper substrate (l-methionine and polyphosphate). By combination of the reaction system optimization and S-adenosyl-methionine regeneration, the titer of hydrogenobyrinic acid was improved from 0.61 to 29.39 mg/L in a 12 h reaction period, representing an increase of 48.18-fold, raising an efficient and rapidly evolutional alternative method to produce high-value-added compounds and intermediate products.