Homologous recombination-mediated genomic editing is urgently needed to obtain high-performance chassis of electroactive microorganisms. However, the existing tools cannot meet the requirement of ...genome-wide editing in Shewanella oneidensis. Here, we develop different CRISPR-Cas systems that are ideal to be employed in AT-rich sequences as the supplements to Cas9. AsCpf1 and BhCas12b show low cell toxicity and superior ability to target sequences and are thus screened out in S. oneidensis MR-1. The PAMs of AsCpf1 and BhCas12b are 5′-TTTV-3′ and 5′-ATTN-3′. For gene deletion, ∼1-kb gene is knocked out and the editing efficiency is 41.67% by BhCas12b-mediated system. For gene replacement, endogenous promoter of nagK was substituted to a constitutive promoter with the efficiency of 25% through BhCas12b system. For gene insertion, the integration efficiency was up to 94.4% and 83.9% via CRISPR-BhCas12b and AsCpf1 tools. This study implies a great potential of CRISPR-BhCas12b/AsCpf1 systems recognizing AT-rich PAMs for genomic editing in S. oneidensis to facilitate multifaceted gene manipulation.
Extracellular electron transfer (EET) of electroactive microorganisms (EAMs) is the dominating factor for versatile applications of bio-electrochemical systems. Shewanella oneidensis MR-1 is one of ...the model EAMs for the study of EET, which is associated with a variety of cellular activities. However, due to the lack of a transcriptional activation tool, regulation of multiple genes is labor-intensive and time-consuming, which hampers the advancement of improving the EET efficiency in S. oneidensis. In this study, we developed an easily operated and multifunctional regulatory tool, that is, a simultaneous clustered regularly interspaced short palindromic repeats (CRISPR)-mediated transcriptional activation (CRISPRa) and interference (CRISPRi) system, for application in S. oneidensis. First, a large number of activators were screened, and RpoD (σ70) was determined as the optimal activator. Second, the effective activation range was identified to be 190–216 base upstream of the transcriptional start site. Third, up- and downregulation was achieved in concert by two orthogonal single guide RNAs targeting different positions. The activation of the cell division gene (minCDE) and repression of the cytotoxic gene (SO_3166) were concurrently implemented, increasing the power density by 2.5-fold and enhancing the degradation rate of azo dyes by 2.9-fold. The simultaneous CRISPRa and CRISPRi system enables simultaneous multiplex genetic regulation, offering the potential to further advance studies of the EET mechanism and application in S. oneidensis.
Electroactive biofilm plays a crucial rule in the electron transfer efficiency of bio-electrochemical systems (BES). However, the slow rate of interfacial electron transfer (IET) restricts practical ...applications of various BES. Here, a modular engineering strategy was developed to enhance IET rate. Firstly, to accelerate the low transmembrane electron transfer rate caused by insulative cell membrane of Shewanella oneidensis, pili-based artificial conductive nanowires and outer-membrane c-cytochrome OmcF from Geobacter sulfurreducens were heterologously expressed to construct transmembrane electron conduits. Secondly, to improve the low electron transfer rate from S. oneidensis to anode owing to the poor electron collection ability of the anode, N-doped carbon nanotubes and polyriboflavin were used to increase the surface area and active sites of the anode. Thirdly, to further reduce the resistance due to the low conductivity of biofilm, the polydopamine was coated in situ to construct high-speed conductive networks, obtaining an unprecedented power density of 5233.7 ± 364.7 mW/m2, ∼83.1-fold higher than that of the wild-type strain (62.2 ± 5.5 mW/m2), and the maximum coulomb efficiency of ESR1 @PDA was 85.4%, which, to the best of our knowledge, is one of the highest output power densities and coulomb efficiencies that have ever reported in the genetically engineered Shewanella strains. This study demonstrated an integrated biotic-electrode modular engineering strategy to boost power generation of electroactive biofilm via synthetic biology and material engineering.
Display omitted
•Modular engineering strategy accelerated interfacial electron transfer rate.•Electron transmembrane channel was constructed by e-pili and outer-membrane c-Cyts.•N-CNT and PRF improved electron collection ability of the anode.•PDA was in situ polymerization on cell surface to form highly conductive networks.•An unprecedented maximum power density of 5233.7 mW/m2 was achieved.
Electroactive microorganisms (EAMs) could utilize extracellular electron transfer (EET) pathways to exchange electrons and energy with their external surroundings. Conductive cytochrome proteins and ...nanowires play crucial roles in controlling electron transfer rate from cytosol to extracellular electrode. Many previous studies elucidated how the c‐type cytochrome proteins and conductive nanowires are synthesized, assembled, and engineered to manipulate the EET rate, and quantified the kinetic processes of electron generation and EET. Here, we firstly overview the electron transfer pathways of EAMs and quantify the kinetic parameters that dictating intracellular electron production and EET. Secondly, we systematically review the structure, conductivity mechanisms, and engineering strategies to manipulate conductive cytochromes and nanowire in EAMs. Lastly, we outlook potential directions for future research in cytochromes and conductive nanowires for enhanced electron transfer. This article reviews the quantitative kinetics of intracellular electron production and EET, and the contribution of engineered c‐type cytochromes and conductive nanowire in enhancing the EET rate, which lay the foundation for enhancing electron transfer capacity of EAMs.
Production of monoclonal antibodies (mAbs) receives considerable attention in the pharmaceutical industry. There has been an increasing interest in the expression of mAbs in Escherichia coli for ...analytical and therapeutic applications in recent years. Here, a modular synthetic biology approach is developed to rationally engineer E. coli by designing three functional modules to facilitate high‐titer production of immunoglobulin G (IgG). First, a bicistronic expression system is constructed and the expression of the key genes in the pyruvate metabolism is tuned by the technologies of synthetic sRNA translational repression and gene overexpression, thus enhancing the cellular material and energy metabolism of E. coli for IgG biosynthesis (module 1). Second, to prevent the IgG biodegradation by proteases, the expression of a number of key proteases is identified and inhibited via synthetic sRNAs (module 2). Third, molecular chaperones are co‐expressed to promote the secretion and folding of IgG (module 3). Synergistic integration of the three modules into the resulting recombinant E. coli results in a yield of the full‐length IgG ≈150 mg L−1 in a 5L fed‐batch bioreactor. The modular synthetic biology approach could be of general use in the production of recombinant mAbs.
There has been an increasing interest in the expression of monoclonal antibodies (mAbs) in Escherichia coli for analytical and therapeutic applications. In this study, three modules are developed to engineer E. coli via pyruvate metabolic engineering, protease engineering, and chaperone engineering. The modular synthetic biology approach is promising for engineering microbial chassis to enhance the production of mAbs.
Display omitted
Human immunodeficiency virus (HIV) infection is the fifth most common cause of death and many new HIV infections occur every year. The prevalence of HIV also seriously affects the ...quality of a patient’s life. More than forty anti-HIV drugs have been put into clinical uses, many of which are chiral molecules with multiple stereogenic centers, for example abacavir, lamivudine, zidovudine, stavudine, tenofovir, atazanavir. However, the chemical synthesis of these chiral intermediates have the disadvantages of low enantiomeric purity and complex synthetic steps. The benefits of asymmetric biosynthesis of chiral drugs include high enantiomeric excess (e.e.), good product selectivity, mild reaction conditions, and less side effects. The biosynthesis of the chiral intermediates of these anti-HIV drugs is thus particularly important. Herein, we review the different sources of enzymes and microbial cells for the asymmetric biosynthesis of the above chiral anti-HIV drug intermediates. We also review recent biotechnology progress in engineering these enzymes and microbial cells with improved biocatalytic activities, including enzyme and cell immobilization, surface display of enzymes, and directed evolution of enzymes. These biotechnology processes enable the efficient biosynthesis of these chiral intermediates, facilitating the industrial production of anti-HIV drugs with reduced costs.
Shewanella oneidensis MR-1, as a model electroactive microorganism (EAM) for extracellular electron transfer (EET) study, plays a key role in advancing practical applications of bio-electrochemical ...systems (BES). Efficient genome-level manipulation tools are vital to promote EET efficiency; thus, a powerful and rapid base editing toolbox in S. oneidensis MR-1 is developed. Firstly a CRISPR/dCas9-AID base editor that shows a relatively narrow editing window restricted to the "-20 to -16" range upstream of the protospacer adjacent motif (PAM) is constructed. Cas9 is also confined by its native PAM requirement, NGG. Then to expand the editable scope, the sgRNA and the Cas-protein to broaden the editing window to "-22 to -9" upstream of the PAM are engineered, and the PAM field to NNN is opened up. Consequently, the coverage of the editable gene is expanded from 89% to nearly 100% in S. oneidensis MR-1. This whole genome-scale cytidine deaminase-based base editing toolbox (WGcBE) is applied to regulate the cell length and the biofilm morphology, which enhances the EET efficiency by 6.7-fold. WGcBE enables an efficient deactivation of genes with full genome coverage, which would contribute to the in-depth and multi-faceted EET study in Shewanella.
The production of the aglycosylated immunoglobulin G (IgG) in
has received wide interest for its analytical and therapeutic applications. To enhance the production titer of IgG, we first used ...synthetic sRNAs to perform a systematical analysis of the gene expression in the translational level in the glycolytic pathway (module 1) and the tricarboxylic acid (TCA) cycle (module 2) to reveal the critical genes for the efficient IgG production. Second, to provide sufficient amino acid precursors for the protein biosynthesis, amino acid biosynthesis pathways (module 3) were enhanced to facilitate the IgG production. Upon integrated engineering of these genes in the three modules (module 1,
; module 2,
and
; module 3,
) and optimization of fermentation conditions, the recombinant
enabled a titer of the full-assembled IgG of 4.5 ± 0.6 mg/L in flask cultures and 184 ± 9.2 mg/L in the 5 L high cell density fed-batch fermenter, which is, as far as we know, the highest reported titer of IgG production in recombinant
.
PDF
