Test strains and plasmids
For the strains and plasmids used in this study, see Additional file 2: Table S6.
Test strain culture
The test strains were cultured, activated, and preserved on activated medium. The components of the activated medium were bovine heart extract powder (10 g/L), casein peptone (10 g/L), and sodium chloride (5 g/L). The medium had a pH value of 7.0. After culturing at 28 ℃ for 36 h, vigorously growing colonies were isolated and observed.
VB12-producing strains were cultured in liquid shake flasks (500 mL conical flasks, 200 mL medium per bottle). See Additional file1: Supplementary Methods 1 for the components of the fermentation medium. The strains in which each gene was episomally expressed and the strains in which each gene was integrated and expressed were cultured in No. 3 medium (Supplementary methods 1). The strains in which genes were episomally expressed were cultured with 50 µg/mL gentamicin, the strains in which the cobA gene were integrated and expressed were cultured with 25 µg/mL chloramphenicol, the strains in which cobT was integrated and expressed were cultured with 50 µg/mL apramycin, and the strains in which cobA and cobT were integrated and expressed were cultured with 25 µg/mL chloramphenicol and 50 µg/mL apramycin. The incubation temperature was 28°C, and the flasks were shaken for 120 h prior to HPLC analysis.
Fermentation sample processing and VB12 detection
The strain fermentation medium was autoclaved for 30 min. After treatment, the samples were centrifuged at 12000 r/min for 5 min, and the supernatant was filtered through a 0.22-µm filter prior to HPLC analysis. Hydroxocobalamin was accurately weighed and dissolved in ultrapure water to prepare a 100 mg/L standard (note: VB12 produced by bacterial fermentation is mainly adenosylcobalamin; adenosylcobalamin is unstable and can convert to hydroxocobalamin when exposed to light, and therefore, the standard is hydroxocobalamin). Standards with concentrations of 10, 25, 50, and 100 mg/L were prepared the analysis; each sample was measured three times, and the average value was taken to make a standard curve. A WATERS 2695 high-performance liquid chromatography system coupled with a W2998 UV detector and a chromatographic column (GL InerSustain C18, 5 µM, 4.6 mm×250 mm) was used. The settings were as follows: column temperature, 30°C; injection volume, 10 µl; detection wavelength, 351 nm; flow rate, 1 mL/min; mobile phase A, acetonitrile; mobile phase B, sodium acetate buffer (pH 3.6); and elution conditions, from 0 to 5 min, 10% acetonitrile gradient elution; from 5 to 10 min, 10–30% acetonitrile gradient elution.
Whole genome sequencing and functional annotation of the test strains
The TIANGEN whole genome DNA extraction kit was used to extract the genomic DNA of the test strains. Purified DNA samples were sent to the ONT platform of Majorbio (Shanghai, China) for Nanopore whole genome sequencing (third-generation whole genome sequencing), and the whole genome sequences of the test strains S305 and Casida A were obtained. Encoding genes, repeats, and noncoding RNAs were predicted for the whole genome DNA sequence of the test strains. The amino acid sequences of the genes were aligned with those in the GO (Gene Ontology), KEGG (Kyoto Encyclopedia of Genes and Genomes), and COG (Cluster of Orthologous Groups of Proteins) databases, and the gene and protein sequences were functionally annotated. Mauve software was used to compare the whole genome DNA sequences of the test strains with the genome sequences of other similar strains published in NCBI to obtain the collinearity of the genomes (Version 2.4.0, the software website http://darlinglab.org/mauve/mauve.html) [35].
Transcriptome sequencing and data analysis
The test strains were cultured in No. 3 medium in shake flasks. Ten millilitres of medium was obtained after culture for 12 h, 24 h, 48 h, and 72 h and centrifuged at 4°C. The pellet was then transferred to a 1.5-ml sterile cryopreservation tube (repeated 3 times) and sent to Majorbio (Shanghai, China) for 2*150 bp/300 bp sequencing on an Illumina HiSeq sequencing platform. To ensure the reliability of subsequent results, the quality-controlled raw data, namely, clean data (reads), were compared with the reference genomes to obtain mapped data (reads) for subsequent analysis. Unqualified reads in the raw data were filtered, and the filtered clean reads were used for subsequent analyses. RSEM software was used to quantitatively analyse the expression levels of the genes, and the quantitative index was FPKM (fragments per kilobase per million reads); that is, in each million sequences, each gene was measured in one thousand bases, and the number of reads was aligned. After obtaining the read counts of genes, differential expression analysis of genes between samples was performed, and p-adjust < 0.05 and |log2FC| ≥ 1 were used as the thresholds for identifying DEGs between samples. Functional enrichment analysis was performed on the gene set, and KEGG pathway enrichment analysis was performed on the genes in the gene set using R script.
Determination of relative gene expression
At similar stages of growth, a Tiangen RNA prep Pure kit to extract the total RNA from the two strains, and cDNA was synthesized using a reverse transcription kit (Takara Biological Company). Primer 5.0 software was used to design primers for the key enzyme genes of the B12 synthesis pathway and the internal reference gene 16S rRNA for the two strains of bacteria. The qPCR primer sequences are shown in Additional file 2: Table S7 and were synthesized by Shanghai Sangon Biological Company. 16S rDNA was used as the internal reference gene. The reaction volume was 20 µL, and the reaction conditions were as follows: predenaturation at 95°C for 30 s and 40 cycles of denaturation at 95°C for 5 s and extension at the appropriate Tm (annealing temperature of the corresponding gene) for 30 s. The cycle threshold was recorded. The relative expression of each gene was calculated using the 2-△△T relative quantitative method.
Episomal overexpression plasmid construction and transformation
Primers were designed (Additional file 2: Table S7), and the construction of the overexpression plasmids for all target genes was basically the same as that for the cobT gene, described below. The cobT gene was amplified using the Casida A strain genome as the template and cobT-28a-F and cobT-28a-R as the primers. The sequence fragment (783 bp) containing gentamicin resistance was amplified using the pJQ200SK plasmid as the template and Gm-28a-F and Gm-28a-R as the primers. The backbone plasmid fragment 1 (719 bp) was amplified using the pET-28a plasmid as the template and P-28a-1F and P-28a-1R as the primers. The backbone plasmid fragment 2 (3798 bp) was amplified using P-28a-2F and P-28a-2R as the primers. The above fragments were assembled by OE-PCR to obtain the pET28a-Gmr-cobT plasmid (see Additional file 1: Figure S7 for the schematic diagram of plasmid construction). Other gene overexpression plasmid construction steps are described in Additional file 1: Supplementary Methods 2. The successfully constructed overexpression plasmid T was transformed into Top10 competent cells; the cells were spread onto a plate containing 50 µg/ml gentamicin and cultured overnight at 37°C. Monoclonal strains were placed into gentamicin-containing LB liquid medium in shake flasks and cultured overnight at 37°C. Plasmids were extracted with a Tiangen DP103 TIANprep Mini Plasmid Kit, and the plasmids were transformed into Casida A competent cells, which were cultured on a plate containing 50 µg/ml gentamycin at 28°C for 36 h, after which monoclonal strains were selected. The successfully overexpressed positive clones were named Casida A-cobA/OP, Casida A-cobT/OP, Casida A-hemA/OP, Casida A-cobJ/OP, Casida A-cobN/OP, Casida A-cobR/OP, and Casida A-cobP/OP.
Integrated expression plasmid construction and transformation
Primers were designed (Additional file 2: Table S7), and the construction of all integrated expression plasmids was basically the same as that for the cobT gene, described below. Using the S305 strain genome as the template, the cobT gene fragment (1017 bp) was amplified with the primers cobT-200SK-F and cobT-200SK-R, the fragment (724 bp) containing the upstream homology arm (600 bp) was amplified with the primers UP-T200SK-F and UP-T200SK-R, the fragment (118 bp) containing the strong promoter ibpA was amplified with the primers pibpA-T200SK-F and pibpA-T200SK-R, and the fragment (498 bp) containing the downstream homology arm (467 bp) was amplified with the primers Down-T200SK-F and Down-T200SK-R. The gene sequence (1009 bp) containing the apramycin resistance gene (Apr) was amplified using the pCRISPomyces-2 plasmid as the template and the primers Apr-200SK-F and Apr-200SK-R. The backbone plasmid fragment (5551 bp) containing the sucrose lethal gene and the gentamicin resistance gene was amplified using the pJQ200SK plasmid as the template and the primers P-200SK-F and P-200SK-R. The above fragments were assembled by OE-PCR to obtain the pSK-cobT-Apr plasmid (see the Additional file 1: Figure S7 for the schematic diagram of plasmid construction). The construction steps for the cobA integrated expression recombinant plasmid pSK-cobA-Cmr are shown in Additional file 1: Supplementary methods 2. The integrated expression plasmid was transformed into Top10 competent cells, which were spread onto a plate containing 50 µg/ml gentamicin and cultured at 37°C overnight. Monoclonal strains were placed into gentamicin-containing LB liquid culture medium in shake flasks and cultured at 37°C overnight. A Tiangen DP103 TIANprep Mini Plasmid Kit was used to extract plasmids, which were transformed into S305 competent cells. Specifically, 200 ng of plasmid was added to competent cells; the cells were shaken gently and then placed on ice for 30 minutes, after which they were heat shocked at 42°C for 90 s, and again placed on ice for 3 min. LB medium (600 mL) at 28°C was added to the cells, which were incubated at 150 r/min for 1 h and then spread on Gmr (50 µg/ml) + Apr (50 µg/ml) LB solid medium and cultured at 28°C until single colonies formed. The primary recombinant bacteria were screened by bacterial resistance to antibiotics in the LB plate, and single clones were picked for subculture for a second homologous recombination. The screened primary recombination-positive single clones were transferred into Gmr (50 µg/ml) + Apr (50 µg/ml) + 15% sucrose liquid medium for three passages at 28°C, and 1 µL of bacterial solution was diluted 100 times and streaked on an Apr (50 µg/ml) + 15% sucrose plate and cultured at 28°C until single colonies formed. A single colony with good growth was picked for PCR using the primers cobT-0651-F and cobT-0651-R to identify the recombinant mutant strain, and the successfully identified mutant strain was named S305-cobA/RC. The transformation of the S305 strain with the pSK-cobT-Apr plasmid followed the same steps as described in the previous section; however, the screening antibiotic apramycin (50 µg/ml) was replaced with the screening antibiotic chloramphenicol (25 µg/ml). The recombinant mutant strains were identified by PCR using the primers cobA-1218-F and cobA-1218-R, and the successfully identified mutant strain was named S305-cobT/RC. Finally, the pSK-cobT-Apr plasmid was transformed into the S305-cobA/RC mutant strain for screening and identification, and the successfully identified mutant strain was named S305-cobA + cobT/RC.
Determination of the growth status of recombinant strains
The wild-type Casida A strain, the overexpression mutants Casida A-cobA/OP, Casida A-cobT/OP, Casida A-hemA/OP, Casida A-cobJ/OP, Casida A-cobN/OP, Casida A-cobR/OP, and Casida A-cobP/OP, and the integrated expression mutants S305-cobA/RC, S305-cobT/RC, and S305-cobA + cobT/RC were separately inoculated in 100 mL of activated culture with corresponding antibiotics at an inoculum dose of 2% and cultured at 28°C and 150 r/min for 24 h (logarithmic growth phase); the bacteria were then transferred to 100 mL of No. 3 medium with corresponding antibiotics and cultured at 28°C and 150 r/min for 168 h. Samples were taken every 12 h, and absorbance was measured 3 times after 10-fold dilution (the wild-type strain was used as the control). The obtained data were analysed to draw growth curves for the strains.