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

(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]