Dna polymerase

The strains and plasmids used in this study are listed in Table 1. E. coli strain DH5α was purchased from Transgene Bio (Transgene Biotechnology Co. Ltd. Beijing, China) for gene cloning. For IPA biosynthesis, E. coli strains W3110, mutant Sun21, and plasmid pSUFAQ were kindly donated by Professor Sheng Yang (Key Laboratory of Synthetic Biology, Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, China). Strains were maintained as glycerol stocks at −80 °C. The IPA sample was purchased from Sigma. All the other chemicals were purchased from Sangon Bio (Sangon Biotechnology Co. Ltd. Shanghai, China). Restriction endonucleases, DNA polymerases, and T4 DNA ligase were purchased from Takara Bio (Takara Biotechnology Dalian Co. Ltd., China) or Sangon Bio.

The oligonucleotides used in this work were synthesized by Sangon Biotech Ltd. (Shanghai, China) and listed in Table S2. PCR amplification was performed following the standard protocols. DNA polymerases were purchased from TaKaRa Co. (Dalian, China); T4 DNA ligases and the restriction endonucleases were purchased from Thermo Fisher Scientific Inc. (Waltham, MA, USA). The Xba I/Bam HI-treated PCR fragments and vector frameworks were ligated with T4 DNA ligase and transformed into E. coli DH5α competent cells. The transformants were screened on the LB plates with gentamicin and the positive recombinant plasmids were verified by DNA sequencing. The sequencing of DNA fragments and plasmid DNAs were performed with an ABI 3730 xl DNA Analyzer sequenator from Thermo Fisher Scientific Inc. (Waltham, MA, USA) using the standard DNA analysis buffer system at Sangon Biotech Ltd. (Shanghai, China); the fragment sizes were then analyzed using the Peakscanner software (Thermo Fisher Scientific). The chemical transformation and the electroporation-mediated transformation were used to transform plasmids into E. coli strains and Pseudomonas strains. The genome DNA, plasmid DNA, and PCR fragments were obtained with the TIANamp Bacteria DNA Kit, TIANprep Mini Plasmid Kit, and TIANquick Mini Purification Kit, respectively (Tiangen, Beijing, China).

Restriction enzymes were purchased from New England Biolabs (Beijing, China) and DNA polymerases were purchased from Takara Bio Inc. (Dalian, China). T4 DNA ligase was purchased from Life Technologies (Shanghai, China). Oligonucleotides and the promoter P_cp628 (link) were synthesized by Life Technologies (Shanghai, China). 1-Deoxynojirimycin (1-DNJ) was purchased from Carbosynth Limited (Berkshire, UK). D-mannosamine hydrochloride, α-mannosidase (from Canavalia ensiformis), 4-nitrophenyl-α-D-mannoside, trehalose and trehalase (from porcine kidney) were purchased from Sigma-Aldrich (St. Louis, USA). The strain Bacillus atrophaeus (Agricultural Culture Collection of China, ACCC 02297) was purchased as a 1-DNJ-producing strain.
All bacteria were routinely grown in Luria-Bertani (LB) medium. The antibiotics ampicillin (100 μg·mL⁻¹), kanamycin (50 μg·mL⁻¹) and apramycin (50 μg·mL⁻¹) were used when necessary.

All reagents were obtained from Sigma‐Aldrich (Saint Louis, MO, USA). Restriction enzymes, DNA polymerases, T4 DNA ligases, DNAse and alkaline phosphatase were obtained from Takara (Kusatsu, Japan), ThermoFisher (Waltham, MA, USA), Roche (Penzberg, Germany) and New England Biolabs (Ipswich, MA, USA). Gel purification and Maxi and miniprep kits were obtained from Qiagen (Hilden, Germany).

The plasmids and primers used in this study are listed in Table 2 and Table 3. Temperature‐sensitive plasmid pKSV7 is a shuttle vector for E. coli and Bacillus, which is stable at 30°C or below and unstable at 37°C or above. Counter‐selective plasmid pKSU is a derivative of pKSV7 that carries the upp gene from B. subtilis 168. Sequences up‐ and downstream of the target gene clusters were PCR amplified and spliced in a subsequent overlapping PCR. The resulting homologous arms were digested with BamHI and SalI and ligated in the same restriction sites of pKSU to yield deletion plasmids. DNA polymerases, restriction enzymes, and T4 DNA ligase were purchased from Takara (Dalian, China). PCR, enzyme digestion, and ligation reactions were performed as recommended by the enzyme suppliers. The DNA fragments were analyzed on 0.8% agarose gels and purified using an Axygen gel DNA recovery kit (Axygen, CA, USA). Deletion plasmids were treated with Bam HI methyltransferase (New England Biolabs, MA, USA) before transformed into B. amyloliquefaciens strains.
Competent E. coli cells were purchased from Transgen Biotech (Beijing, China) and transformed according to the manufacturer's instructions. Deletion plasmids were transformed into B. amyloliquefaciens strains using the high osmolarity electroporation method, with modifications, as described previously (Zhang et al., 2014).

The M. smegmatis and E. coli strains and plasmids used in this study are shown in Table S1. The E. coli strain DH5α was used for cloning. E. coli strains were grown on LB broth agar or in LB broth, 37 °C, 200 rpm. M. smegmatis mc² 155 was grown in 7H9 liquid medium (Difco) supplemented with 0.05% w/v Tween 80, 0.5% glycerol, and 0.5% glucose or was grown on 7H10 agar supplemented with 1% glycerol and 0.5% glucose. Restriction enzymes, T4 DNA ligases, and DNA polymerases were purchased from Takara. Ampicillin, kanamycin, hygromycin were bought from Sangon Biotech Co., whose stock solutions were freshly prepared and filter sterilized. When required, the following antibiotics were used at the final concentration: Ampicillin, 100 μg/mL; kanamycin, 500 μg/mL for E. coli or 200 μg/mL for M. smegmatis; hygromycin, 50 μg/mL. All cultures were incubated at 37 °C.