Bacterial strains and growth conditions
The bacterial strains and plasmids used in this study are listed in Table 1. E. coli XL1-Blue was used as the host for gene cloning and plasmid maintenance, and C. glutamicum ATCC 13032 was used as the main host for gene expression and PHB production. For gene expression in C. glutamicum strains, the plasmids pCES208, E. coli-C. glutamicum shuttle vector was used as the main plasmid [31]. PCR was performed using a C1000™ Thermal Cycler (Bio-Rad, Hercules, CA, USA) with PrimeSTAR HS polymerase (TaKaRa Bio Inc., Shiga, Japan). The nucleotide sequences of all the primers used in this study are listed in Additional file 2: Table S1.
Table 1
Bacterial strains and plasmids used in this study
Strain | Relevant Characteristics | Ref. or Source |
XL1-Blue | recA1 endA1 gyrA96 thi-1 hsdR17 supE44 relA1 lac [F´ proAB lacIqZ∆M15 Tn10 (Tetr)] | Stratagenea |
C. glutamicum | Wild type | ATCC 13032 |
Plasmids | Relevant Characteristics | Ref. or Source |
pCES208 | E. coli-C. glutamicum shuttle vector; KmR | [43] |
pCG-H36A | pCES208 derivative; PH36, Signal sequence of cg1514 | [14] |
pCES-H36-GFP | pCES208 derivative; PH36, GFP | [12] |
pCES208-SapI | pCES208 derivative; two additional SapI restriction enzyme sites | This study |
pA | pCES208 derivative; PH36, phaA from R. eutropha, rrnB T1T2 terminator | This study |
pHA | pCES208 derivative; PH36, phaA from R. eutropha with N-terminal 6×His tag, rrnB T1T2 | This study |
pUHA | pCES208 derivative; PH36, T7 g10 RBS, phaA from R. eutropha with N-terminal 6×His tag, rrnB T1T2 | This study |
pB | pCES208 derivative; PH36, phaB from R. eutropha, rrnB T1T2 terminator | This study |
pHB | pCES208 derivative; PH36, phaB from R. eutropha with N-terminal 6×His tag, rrnB T1T2 terminator | This study |
pUHB | pCES208 derivative; PH36, T7 g10 RBS, phaB from R. eutropha with N-terminal 6×His tag, rrnB T1T2 | This study |
pC | pCES208 derivative; PH36, phaC from R. eutropha, rrnB T1T2 terminator | This study |
pHC | pCES208 derivative; PH36, phaC from R. eutropha with N-terminal 6×His tag, rrnB T1T2 terminator | This study |
pUHC | pCES208 derivative; PH36, T7 g10 RBS, phaC from R. eutropha with N-terminal 6×His tag, rrnB T1T2 | This study |
pABC | pCES208 derivative; PH36-T7 g10 RBS-phaA with N-terminal 6×His tag-rrnB T1T2; PH36-T7 g10 RBS-phaB with N-terminal 6×His tag-rrnB T1T2; PH36-T7 g10 RBS-phaC with N-terminal 6×His tag-rrnB T1T2 | This study |
pABC-S | pABC derivative; PH36-BCD2-fbp-rrnBT1T2; PH36-BCD2-acnR-rrnBT1T2; PH36-BCD2-mez-rrnBT1T2 | This study |
Glu#6 | pCES208 derivative; PI64-BCD21-fbp-rrnBT1T2; PI64-BCD21-acnR-rrnBT1T2; PH36-BCD8-mez-rnBT1T2 | This study |
Fru#1 | pCES208 derivative; PH17-BCD8-fbp-rrnBT1T2; PL10-BCD21-acnR-rrnBT1T2; PH30-BCD8-mez-rrnBT1T2 | This study |
aStratagene, La Jolla, CA, USA |
For plasmid preparation, E. coli was cultivated in Luria-Bertani broth (tryptone, 10 g/L; yeast extract, 5 g/L; and NaCl, 10 g/L NaCl) at 37°C. For cultivation of C. glutamicum strains, BHI (Difco Laboratories, Detroit, MI, USA) and minimal medium (3 g/L K2HPO4, 1 g/L KH2PO4, 2 g/L urea, 10 g/L (NH4)2SO4, 2 g/L MgSO4, 200 µg/L biotin, 5 g/L thiamine, 10 g/L MnSO4, 1 g/L ZnSO4, and 10 mg/L CaCl2) were used with 20 g/L sugar carbon substrate. C. glutamicum cells were inoculated into BHI medium and grown at 30°C for 24 h. Aliquots (500 µL) were transferred into 100 mL minimal media in 250-mL Erlenmeyer flasks and grown at 30°C for 36 h. Kanamycin (Km, 25 µg/L) was added to the culture medium as the sole antibiotic.
Plasmid manipulation and library construction
A graphical summary of plasmid and library construction is provided in Additional file 1: Figure S1. For the expression of three PHB biosynthesis genes (phaA, phaB, and phaC) from R. eutropha, each gene was amplified from the chromosome of R. eutropha by PCR using primer pairs PhaA-F and PhaA-R, PhaB-F and PhaB-R, and PhaC-F and PhaC-R, respectively. After digestion with BamHI and NotI, the PCR products (PhaA, PhaB, and PhaC) were cloned into pCG-H36A [14] between the strong H36 synthetic promoter and rrnBT1T2 terminator to yield pA, pB, and pC, respectively. To add a 6⋅His tag (HHHHHH) to the N-terminus of PhaA, PhaB, and PhaC, each gene was amplified from the R. eutropha chromosome by PCR using primer pairs H-PhaA-F and PhaA-R, H-PhaB-F and PhaB-R, and H-PhaC-F and PhaC-R, respectively. After digestion with BamHI and NotI, the PCR products were cloned into pCG-H36A [14] to yield pHA, pHB, and pHC, respectively. To introduce the T7 gene10 RBS sequence to His-tagged phaA, phaB, and phaC, each gene was PCR amplified from the R. eutropha chromosome with the primer pairs UH-PhaA-F and PhaA-R, UH-PhaB-F and PhaB-R, and UH-PhaC-F and PhaC-R, respectively. After digestion with BamHI and NotI, the PCR products were cloned into pCG-H36A [14] to yield pUHA, pUHB, and pUHC, respectively.
The pCES208-SapI plasmid is a derivative of the pCES208 shuttle vector with two additional SapI-Type IIS restriction enzyme sites. For the introduction of SapI restriction enzyme sites, a PCR fragment was amplified by PCR using the primer pair SapI-F and SapI-R. After digestion with NcoI, the PCR product was cloned into pCES208 to obtain pCES208-SapI. For construction of whole PHB synthesis pathway by combining the expression systems for phaA, phaB, and phaC from R. eutropha, which are all composed of H36 synthetic promoter, T7 gene10 RBS, N-terminal 6⋅His Tag, and rrnBT1T2 terminator, each expression system was amplified from pUHA, pUHB, and pUHC by PCR with primer pairs, A-BsaI-F and A-BsaI-R, B-BsaI-F and B-BsaI-R, and C-BsaI-F and C-BsaI-R, respectively. After digestion with BsaI-type IIS restriction enzyme, all PCR products were combined with SalI and NotI-treated pCES208-SapI for the assembly of expression systems for phaA, phaB, and phaC to yield pABC.
For the expression of the fructose biphosphatase (fbp), TetR-type aconitase repressor (acnR), and malate dehydrogenase (mez) genes from C. glutamicum, each gene was amplified using three different bicistronic design (BCD) of RBSs [38] from the C. glutamicum chromosome by PCR with primer pairs: BCD2-Fbp-F and Fbp-R, BCD21-Fbp-F and Fbp-R, BCD8-Fbp-F and Fbp-R, BCD2-AcnR-F and AcnR-R, BCD21-AcnR-F and AcnR-R, BCD8-AcnR-F and AcnR-R, BCD2-Mez-F and Mez-R, BCD21-Mez-F and Mez-R, and BCD8-Mez-F and Mez-R, respectively. In all reverse primers (Fbp-R, AcnR-R, and Mez-R), the lpp terminator sequence was used to add the sequence at the end of each gene. After digestion with BamHI and NotI, the PCR products were cloned into pCES-H36-GFP [12], which has a strong H36 promoter for the expression of target genes to yield pCES-H36-BCD2-Fbp-T, pCES-H36-BCD21-Fbp-T, pCES-H36-BCD8-Fbp-T, pCES-H36-BCD2-AcnR-T, pCES-H36-BCD21-AcnR-T, pCES-H36-BCD8-AcnR-T, pCES-H36-BCD2-Mez-T, pCES-H36-BCD21-Mez-T, and pCES-H36-BCD8-Mez-T. To introduce 20 synthetic promoters (L10, L26, L80, I9, I12, I15, I16, I29, I51, I64, H3, H4, H5, H17, H28, H30, H34, H36, H43, and H72) previously developed by FACS-based screening of promoter library [12], each promoter was prepared from each clone by KpnI and BamHI digestion. Each fragment was cloned into pCES-H36-BCD2-Fbp-T, pCES-H36-BCD21-Fbp-T, pCES-H36-BCD8-Fbp-T, pCES-H36-BCD2-AcnR-T, pCES-H36-BCD21-AcnR-T, pCES-H36-BCD8-AcnR-T, pCES-H36-BCD2-Mez-T, pCES-H36-BCD21-Mez-T, and pCES-H36-BCD8-Mez-T to yield 60 combinations of promoters and BCDs for the expression of fbp, acnR, and mez genes, respectively. To construct a metabolic network library by combinatorial assembly of the expression systems for fbp, acnR, and mez genes, each pool of 60 combinations for each gene was PCR amplified with the primer pairs Fbp-SapI-F and Fbp-SapI-R, AcnR-SapI-F and AcnR-SapI-R, and Mez-SapI-F and Mez-SapI-R, respectively. After digestion with SapI-Type IIS restriction enzyme, all digested PCR products were assembled into SapI-treated pABC to yield a metabolic network library for PHB production. To construct a control plasmid with the strongest expression systems for all the fbp, acnR, and mez genes from C. glutamicum, in addition to the phaA, phaB, and phaC genes from R. eutropha, expression systems for fbp, acnR, and mez genes, which consist of the H36 synthetic promoter (strongest promoter) and BCD2 (strongest BCD), were PCR amplified with primer pairs Fbp-SapI-F and Fbp-SapI-R, AcnR-SapI-F and AcnR-SapI-R, and Mez-SapI-F and Mez-SapI-R, respectively. After digestion with SapI-Type IIS restriction enzyme, all PCR products were combined with SapI-treated pABC to yield pABC-S.