Vitamin B₁, which is also known as thiamine, was the first B vitamin to be identified. Thiamine pyrophosphate (TPP), the active form of thiamine, can inhibit the activity of cholinesterase, reduce skin inflammation, prevent seborrheic dermatitis, or eczema, and improve skin health. Thiamine biosynthesis results from the coupling of the pyrimidine and the thiazole moieties to form thiamine phosphate (Dorrestein et al., 2004; Jurgenson et al., 2009; Cea et al., 2020). Escherichia coli, Salmonella typhimurium, and Bacillus subtilis are the most thoroughly studied thiamine production organisms (Begley et al., 1999).

In chassis cell S. typhimurium, the thiamine pyrimidine moiety can be produced through de novo purine biosynthesis or independently of the purF gene through the alternative pyrimidine biosynthesis (APB) pathway (Downs and Roth, 1991; Downs, 1992). According to the phenotypic characteristics of the abpA mutant, follow-up studies concluded that the functional APB pathway is essential for thiamine synthesis when S. typhimurium grows in the presence of exogenous purines (Downs and Petersen, 1994). Research has shown that overexpression of thiA, nmtA, and thiP in Aspergillus oryzae can increase the vitamin B₁ yield fourfold compared to the wild-type (Tokui et al., 2011). Based on the riboswitch mechanism, mutations in the genes of thiamine pyrophosphate kinase activity (thiN) and thiamine-related transport proteins (YkoD and YuaJ) were introduced in B. subtilis TH95. It was recently reported that thiamine biosynthesis is strictly regulated by TPP riboswitches in bacteria/eukaryotes and transcriptional repressors in archaea (Hwang et al., 2017). E. coli has emerged as the preferred cell factory for TPP production after a riboswitch-based biosensors enabled the discovery of thiamine transporters, combined with overexpression of the native thiFSGHCE and thiD genes, which are closely related to Fe-S metabolism (Figure 1A and Table 1; Cardinale et al., 2017).

However, thiC/thiH in the thiamine biosynthetic pathway is involved in Fe–S metabolism and is inhibited by S-adenosylmethionine (SAM) metabolites, and the catalytic activity of ThiC enzyme (Figure 2) is very low (k_cat = 0.002 s^–1) which is one of the main metabolic bottlenecks (Palmer and Downs, 2013). In addition, the cost of chemical production of thiamine is very low, and the production of engineered strains needs to be increased to be expected to be industrialized.

Riboflavin is an important precursor of flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD) (Balasubramaniam et al., 2019; Andreieva et al., 2020). Riboflavin insufficiency manifests as persistent anemia (Shi et al., 2014). The biosynthesis of riboflavin begins with guanosine triphosphate and ribose-5-phosphate, followed by six enzymatic steps (Fischer and Bacher, 2005). Burgess et al. (2004) found that overexpression of the ribABCGH genes can increase riboflavin production. Later, it was found that there were both nucleotide substitutions and deletions in the regulatory region of the rib operon. By deregulating the rib operon and purine pathway of B. subtilis, riboflavin production was greatly improved. The specific genetic engineering steps included overexpression of the ribA gene and deletion of the purR gene, after which maximum output of riboflavin reached more than 826.52 mg/L (Figure 1B; Shi et al., 2014). In Candida famata overexpression of sef1 and imh3 was combined with classic mutagenesis methods to construct the high riboflavin-producing strain AF-4. As a result, 1026 ± 50 mg/L of riboflavin can be produced during a fed-batch cultivation in a lab-scale fermenter. This research has made a great contribution to industrial production of riboflavin (Dmytruk et al., 2011, 2014). The two most important industrial producers are Ashbya gossypii and B. subtilis.

In A. gossypii, malate synthase in the glyoxylate cycle is essential for riboflavin production. Deletion of the malate synthase gene (ACR268C) decreased riboflavin production 10-fold compared to the wild-type strain. Conversely, overexpression of the ACR286C gene significantly increased the yield of riboflavin by 70%. These results demonstrated that malate synthase is a new target for improving the production of riboflavin (Sugimoto et al., 2009). Abbas and Sibirny (2011) introduced the icl gene, overexpressed the gly1, prs2,4, and prs3 genes, as well as knocking out the vma4, shm2, and bas1 genes, resulting in riboflavin production of more than 20 g/L.

In B. subtilis, Schwechheimer et al. (2016) overexpressed riboflavin biosynthesis genes, decreased the activity of the flavin kinase RibCF, and improved the de novo purine synthesis and pentose supply, after which the riboflavin yield reached more than 26 g/L. At present, the bottleneck of riboflavin production is mainly due to the poor genetic stability of the engineered strain, and more by-products produced by fermentation, which restrict the high yield of riboflavin.

Niacin is the precursor in the synthesis of the pyridine coenzymes NAD (nicotinamide adenine dinucleotide) and NADP (nicotinamide adenine dinucleotide phosphate) (Chand and Savitri, 2016; Chauhan and Poddar, 2019; Tannous et al., 2020). It is found at relatively high concentrations in internal organs of animal, muscle tissues, and fruits. Currently, niacin is mainly used as a feed additive to increase the utilization of feed protein, or as a pharmaceutical intermediate in the synthesis of various drugs. So far, there is no systematic description of a commercial fermentation process of nicotinic acid (NA) or nicotinamide (NAM). Industrial production methods are mainly ammonia oxidation and electrolytic oxidation, but the former has high production costs and needs to be above 300°C during the reaction, and the latter has low costs of production, however, the efficiency of electrolysis is not high, which limits the industrial production of niacin (Chand and Savitri, 2016).

Recent reports describe the use of recombinant E. coli expressing Rhodococcus rhodochrous nitrile hydratase for vitamin B₃ production. At low cell density, nicotinamide was produced in fed-batch mode, and the product concentration reached 390 g/L. After high-density culture in 5 L bioreactor, the concentration of nicotinamide reached 508 g/L in 60 min (Figure 1D; Wang et al., 2017). Belenky et al. (2011) showed that the disruption of nrt1 results in increased export of nicotinamide riboside (NR). Moreover, disruption of the niacin transporter Tna1 can also increase the output of niacin, revealing that cells regulate the intracellular NAD⁺ metabolic process by balancing the transport of niacin, the precursor of NAD⁺. On the basis of adding 5 mM niacin, yeast cells can produce 8 mg/L nicotinamide mononucleotide (Belenky et al., 2011).

Vitamin B₅, also known as pantothenic acid, is composed of pantoic acid and β-alanine (β-Ala), which is a precursor of coenzyme A (Leonardi and Jackowski, 2007). It plays an important role in maintaining the health of skin and blood. Its general function is to participate in the production of energy in the body, but it can also control the fat metabolism, and is also an essential nutrient for the brain and nerves. There are chemical and microbial synthesis methods for the synthesis of pantothenic acid, whereby microbial methods can be used to directly synthesize optically pure D-pantothenic acid.

Sahm and Eggeling (1999) adopted a series of methods to increase the production of pantothenic acid, including the deletion of the ilvA gene and the overexpression of the ilvBNCD and panBC genes. The pantothenic acid production of the best strain reached 1000 mg/L (Figure 1F; Sahm and Eggeling, 1999). Huser et al. (2005) also used Corynebacterium glutamicum to produce pantothenic acid. They deleted the ilvA gene, inhibited the expression of the ilvE gene and overexpressed the ilvBNCD gene. The final titer of pantothenate reached 1.75 g/L (Huser et al., 2005). Studies have shown that the specific activity of pantothenic acid synthase PanC of C. glutamicum is 205.10 U/mg. Adding substrates (D-pantothenic acid and β-Ala) to E. coli containing the enzyme can be produced 97.10 U/mg within 32 h, the conversion rate of pantothenic acid was 99.10%. However, the reported work had production defects, which required the addition of exogenous substrate pantothenic acid, and the high market price of pantothenic acid seriously restricted the industrialization of this method. Another chassis organism that is commonly used to produce pantothenic acid is B. subtilis. Hohmann et al. (2016) clarified the highest production of pantothenic acid by overexpressing ilvBHCD, panBCDE, serA, and glyA, as well as the enzymes of the glycine cleavage cycle the purpose is to increase the number of precursors for pantothenic acid synthesis (Figure 1F). The maximal output of the best strain reached 82–86 g/L during a 48 h fed-batch fermentation, opening up a new chapter of vitamin production in the biological world (Hohmann et al., 2016).

There are six forms of vitamin B₆, including pyridoxine (PN), pyridoxal (PL), and pyridoxamine (PM), as well as their respective phosphate derivatives. It is a water-soluble vitamin, which exists in the form of phosphate in the body. The most versatile form of vitamin B₆ is pyridoxal 5′-phosphate (PLP), which is a cofactor of many proteins and enzymes in all organisms. As the most widely available commercial form, PN hydrochloride is extensively used in the pharmaceutical and food industries (Eliot and Kirsch, 2004).

Two de novo synthesis routes have been reported the 1-deoxyxylulose 5-phosphate (DXP)-dependent pathway and the DXP-independent pathway (Tanaka et al., 2005). From large-scale screening studies of different strains, found that the Gram-negative bacterium Sinorhizobium meliloti is the best producer of vitamin B₆, reaching a titer of 103 mg/L of B₆ isoforms within 168 h. Vitamin B₆ production was further increased to 1.30 g/L by expressing the E. coli epd gene and the native dxs gene in this S. meliloti strain (Figure 1E; Hoshino et al., 2007). E. coli and B. subtilis were also engineered to produce vitamin B₆. The vitamin B₆ production was enhanced to 78 mg/L within 31 h in E. coli, and B. subtilis produced 65 mg/L of PN when supplied with the precursor 4-hydroxy-L-threonine (4HT) (Hoshino et al., 2004). At present, the industry mainly adopts the oxazole method to produce vitamin B₆, and the current research also focuses on the improvement of the oxazole method synthesis process. In the process of biosynthesis, the PdxJ enzyme activity is very low (k_cat = 0.07 s^–1), and the reaction step catalyzed by this enzyme is the rate-limiting step in the VB₆ biosynthetic pathway. The intermediate metabolite 4-phosphate hydroxy-threonine (4HTP) is cytotoxic and is also the main bottleneck of biosynthesis. Therefore, the fermentative production of vitamin B₆ requires more effort to meet the commercial demand.

Biotin is indispensable for the normal metabolism of fats and proteins (Lin and Cronan, 2011; Selvam et al., 2019). It is a nutrient necessary for human growth, development and normal function. Biotin combines with enzymes to participate in the process of carbon dioxide fixation and carboxylation in the body. The current large-scale production of D-biotin is mainly based on the Sternbach synthetic route, and the current industrial production method were improved on this basis. Unless biosynthetic methods can obtain high output at low cost, it is difficult to shake the position of chemical synthesis technology in industrial production. Nevertheless, it was recently reported that some microorganisms can overproduce biotin, which has been elaborated in C. glutamicum, Mesorhizobium loti, and S. meliloti.

In Agrobacterium and Rhizobium HK40, overexpression of the biotin operon from E. coli driven by the powerful tac promoter and introducing a modified RBS in front of bioB resulted in a biotin yield of 110 mg/L (Figure 1G; Streit and Entcheva, 2003). If the native biotin operon is overexpressed in B. subtilis, most enzymes will be strongly inhibited by the by-product of SAM. However, the high demand for SAM by biotin synthase and 7,8-diaminononanoate synthase is still a bottleneck that must be addressed in future research. If lysine is supplied to B. subtilis, BioK will use lysine as the amino donor of the biotin precursor to promote the production of biotin precursor (dephosphorization biotin), and the fermentation process used carbon-limited fed-batch growth conditions with computer control of dissolved oxygen concentrations, but the maximal titer can only reach 21 mg/L biotin. Therefore, improving the catalytic mechanism of biotin synthase is also a challenge for future research (Van Arsdell et al., 2005; Lin and Cronan, 2011).

Naturally occurring folic acid is mostly found in the form of polyglutamic acid, and the biologically active form of folic acid is tetrahydrofolate (Myszczyszyn et al., 2019). Deficiency can lead to reduced hemoglobin content in red blood cells, impaired cell maturation and megaloblastic anemia (Lucock, 2000). B. subtilis or A. gossypii were successfully engineered to produce folic acid.

Jagerstad and Jastrebova (2013) achieved a 5-methyltetrahydrofolate (THF) titer of 0.95 mg/L by increasing the supply of precursor substances and blocking the catabolic pathway of THF in B. subtilis. With the continuous research progress, A. gossypii has attracted increasing interest as the chassis strain for folic acid production. A. gossypii can synthesize 0.04 mg/L of folic acid naturally, which can reach 6.59 mg/L after metabolic engineering treatment. This is also the highest production value reported to date (Figure 1C; Serrano-Amatriain et al., 2016). Since the commercial chemical synthesis of folic acid is cheap, unless the environmentally unfriendly part of the chemical synthesis process is restricted, there is still a long way to go for the fermentation of this product.

Cobalamin is the only vitamin containing metal elements. Cobalamin is the general term for a class of corrin compounds containing cobalt (Osman et al., 2021). It is the largest and most complex vitamin molecule discovered so far. Vitamin B₁₂ deficiency leads to increased formation of ring sideroblasts in pre-myelodysplastic syndromes (Kitago et al., 2020).

Vitamin B₁₂ is synthesized by microorganisms through de novo synthesis or salvage synthesis in nature, but higher-animals and plants cannot produce it (Figure 1H; Fang et al., 2017). Although in the 19th century, researchers have completed the full chemical synthesis of vitamin B₁₂, the chemical synthesis method is too complicated and expensive, so the world’s major suppliers rely on microbial fermentation to produce vitamins. Pseudomonas denitrificans and Propionibacterium freudenreichii being widely used in industrial fermentation to produce vitamin B₁₂. In order to improve the productivity of vitamin B₁₂, researchers have adopted a random mutagenesis method to construct a vitamin B₁₂ overproducing strains by ultraviolet rays, nitrosoguanidine (NTG), nitrosomethylurethane and ethyleneimine (Blanche et al., 1992, 1995a,b). Propionibacterium shermanii was reported to produce vitamin B₁₂ with a maximum titer of 200 mg/L (Sych et al., 2016). However, the aerobic P. denitrificans remains the most used industrial host, and the effect is obvious. Moreover, P. denitrificans has stronger production capacity than the anaerobic strain that produces vitamin B₁₂ and is widely used in industrial production. Xia et al. (2015) increased the production of vitamin B₁₂ to 198 ± 4.60 mg/L by optimizing the fermentation medium using response surface method. Our research group used E. coli strain MG1655 (DE3) as the starting strain to achieve de novo synthesis of vitamin B₁₂ (Fang et al., 2018). Cai et al. (2018) later used riboswitch elements in S. meliloti for the first time, and successfully developed a flow cytometry high-throughput screening system for high-yield VB₁₂ strains. The vitamin B₁₂ titer of the best strain, S. meliloti MC5–2, reached 156 ± 4.20 mg/L, but the yield was still relatively low. At the same time, they also emphasized that the titer of vitamin B₁₂ is greatly dependent on the medium composition (Cai et al., 2018).