Development of biotin-prototrophic and -hyperauxotrophic Corynebacterium glutamicum strains

Abstract

To develop the infrastructure for biotin production through naturally biotin-auxotrophic Corynebacterium glutamicum, we attempted to engineer the organism into a biotin prototroph and a biotin hyperauxotroph. To confer biotin prototrophy on the organism, the cotranscribed bioBF genes of Escherichia coli were introduced into the C. glutamicum genome, which originally lacked the bioF gene. The resulting strain still required biotin for growth, but it could be replaced by exogenous pimelic acid, a source of the biotin precursor pimelate thioester linked to either coenzyme A (CoA) or acyl carrier protein (ACP). To bridge the gap between the pimelate thioester and its dedicated precursor acyl-CoA (or -ACP), the bioI gene of Bacillus subtilis, which encoded a P450 protein that cleaves a carbon-carbon bond of an acyl-ACP to generate pimeloyl-ACP, was further expressed in the engineered strain by using a plasmid system. This resulted in a biotin prototroph that is capable of the de novo synthesis of biotin. On the other hand, the bioY gene responsible for biotin uptake was disrupted in wild-type C. glutamicum. Whereas the wild-type strain required approximately 1 μg of biotin per liter for normal growth, the bioY disruptant (ΔbioY) required approximately 1 mg of biotin per liter, almost 3 orders of magnitude higher than the wild-type level. The ΔbioY strain showed a similar high requirement for the precursor dethiobiotin, a substrate for bioB-encoded biotin synthase. To eliminate the dependency on dethiobiotin, the bioB gene was further disrupted in both the wild-type strain and the ΔbioY strain. By selectively using the resulting two strains (ΔbioB and ΔbioBY) as indicator strains, we developed a practical biotin bioassay system that can quantify biotin in the seven-digit range, from approximately 0.1 μg to 1 g per liter. This bioassay proved that the engineered biotin prototroph of C. glutamicum produced biotin directly from glucose, albeit at a marginally detectable level (approximately 0.3 μg per liter).

Fig 1

Biotin-biosynthetic pathways and the relevant genes in C. glutamicum. The biotin precursor pimelate thioester is either a CoA derivative or an ACP derivative. The products of the FAS-I-type fatty acid synthetases encoded by fasA and fasB are considered to be acyl-CoAs because the closely related C. ammoniagenes (previously referred to as Brevibacterium ammoniagenes) has been shown to generate CoA derivatives (39). The biotin-biosynthetic pathway of C. glutamicum is incomplete due to the lack of the bioF gene and probably the gene(s) for the de novo synthesis of pimeloyl-CoA (or -ACP). For the synthesis of a pimelate moiety, two different routes have been proposed: the E. coli bioC-bioH route (dotted arrows) and the B. subtilis bioI route (thick gray arrow). Both routes are believed to depend on fatty acid synthesis, but at different levels. In E. coli, BioC catalyzes methylation of malonyl-CoA to form malonyl-CoA methyl ester, which enters the fatty-acid-biosynthetic pathway to generate pimeloyl-ACP methyl ester after two cycles of chain elongation (9). The methyl ester moiety is cleaved by BioH to produce the biotin precursor pimeloyl-ACP (9). In B. subtilis, BioI catalyzes oxidative C-C bond cleavage of long-chain acyl-ACPs to produce pimeloyl-ACP (10). The process of incorporating exogenous pimelic acid into the biotin-biosynthetic pathway remains unclear in C. glutamicum, whereas in B. subtilis, this step is catalyzed by the bioW gene product (32). The uptake of pimelic acid is considered to occur by passive diffusion, as is the case with E. coli and several other bacteria (35). In this study, the E. coli bioBF genes and the B. subtilis bioI gene were introduced into C. glutamicum to establish the biotin prototroph, while the endogenous bioY gene was deleted in C. glutamicum to establish the biotin hyperauxotroph.

Fig 2

Schematic diagram of the creation of strain C. glutamicum BF-3 carrying the E. coli bioBF genes on its genome. The E. coli genomic region comprising the bioBF gene cluster and its promoter/operator sequence (P/O) was cloned into a vector for gene replacement, followed by integration into the noncoding region in the C. glutamicum genome.

Fig 3

Growth of wild-type strain ATCC 13032, strain BF-3, and the pBbioI^gap carrier BFI-4. Cultivations were carried out in biotin-free MM (○) and MM supplemented with 100 mg of pimelic acid (▲) or 1 μg of biotin (■) per liter. The values are means of replicate cultures, which showed <5% differences from each other. OD₆₆₀, optical density at 660 nm.

Fig 4

Growth responses of wild-type strain ATCC 13032 and its bioY-disrupted ΔbioY strain to biotin. (A) Appropriate dilutions (approximately 10³ cells/ml) of cultures were spread onto MM agar plates and cultured at 30°C for 1 day under the indicated biotin concentrations. The images show one representative result from three independent experiments. (B) Cultivations were carried out at 30°C in MM liquid culture with 0 μg (◆), 0.1 μg (■), 1 μg (□), 10 μg (△), 100 μg (▲), and 1,000 μg (●) of biotin per liter. The values are means of replicate cultures, which showed <5% differences from each other.

Fig 5

Bioassays of different concentrations of biotin using the ΔbioB strain and the ΔbioBY strain as indicator strains. (A) The two indicator strains were tested for the ability to form halos on MM agar plates with paper disks supplemented with 100 μl of various concentrations of biotin. The plates were incubated overnight at 30°C. The images show one representative result from three independent experiments. (B) Correlations between biotin concentrations and halo sizes formed by the ΔbioB strain and the ΔbioBY strain. The values are means and standard deviations of three independent experiments.

Fig 6

Biotin-forming ability of strain BFI-4 in agar piece assays. The engineered biotin prototroph BFI-4, as well as wild-type strain ATCC 13032 and strain BF-3, was cultivated on MM agar pieces with and without 100 mg of pimelic acid or dethiobiotin per liter. After cultivation for 7 days, the agar pieces were transferred onto bioassay plates containing the ΔbioB strain as the indicator strain. The plates were incubated overnight at 30°C. The images show one representative result from three independent experiments. Strains ATCC 13032 and BF-3, both biotin auxotrophs, appear to have grown on the biotin-free MM agar pieces with no supplementation, but this was certainly due to the carryover of biotin.