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Table 3 Operational tools of metabolic engineering for clostridia [101]

From: Pathway dissection, regulation, engineering and application: lessons learned from biobutanol production by solventogenic clostridia

Engineering tool Characteristics Applications Refs.
(i) Plasmids for gene overexpression
 pMTL80000 plasmid series (1) A standardized modular E. coli/Clostridium shuttle vector system Allowing reliable stringency and a broad range of inducibility [225]
  (2) Uses native clostridial promoters or inducible promoters to promote plasmid-borne gene expression   [226]
 Bacillus/C. acetobutylicum shuttle vector pFNK1 Harbors the replicon derived from pIM13 and a macrolide lincosamine resistance gene as selection marker Overexpression of acetoacetate decarboxylase (adc) and phosphotransbutyrylase (ptb) genes in C. acetobutylicum ATCC 824 [227]
 Plasmids pAN1 and pAN2 Contains compatible E. coli replicons and antibiotic resistance genes Expressing the ϕ3TI methyltransferase gene from B. subtilis in C. acetobutylicum [92]
 Derivatives of pSOS94 and pHT3 plasmids Gene expression reporter system using the lacZ gene from Thermosulfurogenes EM1 Actively expressing β-galactosidase and phosphotransbutyrylase in C. acetobutylicum ATCC 824 [228]
(ii) Chromosomal integration for gene overexpression
 Cargo technique Manipulates chromosomal content to knockout the targeted gene and integrate large DNA fragments into the chromosome (1) Engineering C. acetobutylicum to convert acetone into isopropanol
(2) Engineering C. acetobutylicum to secrete various synthetic cellulosome components
[229, 230]
(iii) Synthetic untranslated regions for gene overexpression
 Synthetic untranslated regions A short single-stranded 5′ untranslated region (UTR) sequence Short single-stranded UTR decreased gene expression, but addition of a stem–loop at the 5′ end of mRNA increased the levels of mRNA and protein expression [231]
(iv) Integrative plasmids for gene inactivation
 Non-replicative suicide plasmids Homologous recombination into the respective genes Inactivating SolR to improve solventogenic phenotype [232]
 Replicating plasmids Integration into a specific gene to inactivate Using replicating plasmids to inactivate the genes spo0A, sigE, and sigG [233]
 CRISPR–Cas9 system Clustered regularly interspaced short palindromic repeats system Deleting spo0A with an editing efficiency of 100% in C. acetobutylicum [94]
(v) Antisense RNA for gene down-regulation
 Plasmid-encoded asRNA Knocking-out or down-regulating targeted genes Down-regulating spoIIE to prolong and elevate solventogenesis and delay initiation of endospore formation [234]
(vi) Small RNA system for gene down-regulation
 Small regulatory RNA (sRNA)‐based system Consisting of a target recognition site, MicC sRNA scaffold, and an RNA chaperone Hfq Knocking down the pta gene expression in strain PJC4BK to reduced acetic acid production [96]
(vii) ClosTron system and related techniques for gene inactivation
 Group II introns (1) Comprising a multi-domain intron-encoded protein that mediates sequence recognition and self-splicing
(2) Engineered by including a retrotransposition-activated marker providing erythromycin resistance after intron insertion
(3) Another variant, combining group II intron and homologous recombination
(1) Alternating use of erythromycin and thiamphenicol resistance genes for generating various gene knockout mutants including three double- and two triple-knockout strains
(2) An alternative gene deletion method that is very elaborate and time-consuming because of successive transfers and large numbers of colonies that need to be screened to identify positive clones
[235, 236]
(viii) Allelic exchange mutagenesis
 Allele-coupled exchange (ACE) Developed to knock out genes or integrate large DNA fragments into the genome with the selectable phenotype Generating stable deletion mutants of C. acetobutylicum [61, 237, 238]