Riboflavin (RF) and its active forms, the cofactors flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD), have been extensively used in the food, feed and pharmaceutical industries. Modern commercial production of riboflavin is based on microbial fermentation, but the established genetically engineered production strains are facing new challenges due to safety concerns in the food and feed additives industry. High yields of flavin mononucleotide and flavin adenine dinucleotide have been obtained using whole-cell biocatalysis processes. However, the necessity of adding expensive precursors results in high production costs. Consequently, developing microbial cell factories that are capable of efficiently producing flavin nucleotides at low cost is an increasingly attractive approach. The biotechnological processes for the production of RF and its cognate cofactors are reviewed in this article.

2,3-Butanediol (2,3-BD) with low toxicity to microbes, could be a promising alternative for biofuel production. However, most of the 2,3-BD producers are opportunistic pathogens that are not suitable for industrial-scale fermentation. In our previous study, wild-type Bacillus subtilis 168, as a class I microorganism, was first found to generate only d-(−)-2,3-BD (purity >99 %) under low oxygen conditions. In this work, B. subtilis was engineered to produce chiral pure meso-2,3-BD. First, d-(−)-2,3-BD production was abolished by deleting d-(−)-2,3-BD dehydrogenase coding gene bdhA, and acoA gene was knocked out to prevent the degradation of acetoin (AC), the immediate precursor of 2,3-BD. Next, both pta and ldh gene were deleted to decrease the accumulation of the byproducts, acetate and l-lactate. We further introduced the meso-2,3-BD dehydrogenase coding gene budC from Klebsiella pneumoniae CICC10011, as well as overexpressed alsSD in the tetra-mutant (ΔacoAΔbdhAΔptaΔldh) to achieve the efficient production of chiral meso-2,3-BD. Finally, the pool of NADH availability was further increased to facilitate the conversion of meso-2,3-BD from AC by overexpressing udhA gene (coding a soluble transhydrogenase) and low dissolved oxygen control during the cultivation. Under microaerobic oxygen conditions, the best strain BSF9 produced 103.7 g/L meso-2,3-BD with a yield of 0.487 g/g glucose in the 5-L batch fermenter, and the titer of the main byproduct AC was no more than 1.1 g/L. This work offered a novel strategy for the production of chiral pure meso-2,3-BD in B. subtilis. To our knowledge, this is the first report indicating that metabolic engineered B. subtilis could produce chiral meso-2,3-BD with high purity under limited oxygen conditions. These results further demonstrated that B. subtilis as a class I microorganism is a competitive industrial-level meso-2,3-BD producer.

5-Aminolevulinic acid (5-ALA) is the key precursor for the biosynthesis of tetrapyrrole compounds, with wide applications in medicine, agriculture and other burgeoning fields. Because of its potential applications and disadvantages of chemical synthesis, alternative biotechnological methods have drawn increasing attention. In this review, the recent progress in biosynthetic pathways and regulatory mechanisms of 5-ALA synthesis in biological hosts are summarized. The research progress on 5-ALA biosynthesis via the C4/C5 pathway in microbial cells is emphasized, and the corresponding biotechnological design strategies are highlighted and discussed in detail. In addition, the detection methods and applications of 5-ALA are also reviewed. Finally, perspectives on potential strategies for improving the biosynthesis of 5-ALA and understanding the related mechanisms to further promote its industrial application are conceived and proposed.