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Shewanella oneidensis MR-1, as a model exoelectrogen with divergent extracellular electron transfer (EET) pathways, has been widely used in microbial fuel cells (MFCs). The electron transfer rate is largely determined by riboflavin (RF) and c-type cytochromes (c-Cyts). However, relatively low RF production and inappropriate amount of c-Cyts substantially impede the capacity of improving the EET rate. In this study, coupling of riboflavin de novo biosynthesis and c-Cyts expression was implemented to enhance the efficiency of EET in S. oneidensis. First, the upstream pathway of RF de novo biosynthesis was divided into four modules, and the expression level of 22 genes in above four modules was fine-tuned by employing promoters with different strengths. Among them, genes zwf*, glyA, and ybjU which exhibited optimal RF production were combinatorially overexpressed, leading to the enhancement of maximum output power density by 166%. Second, the diverse c-Cyts genes were overexpressed to match high RF production, and omcA was selected for further combination. Third, RF de novo biosynthesis and c-Cyts expression were combined, resulting in 2.34-fold higher power output than the parent strain. This modular and combinatorial manipulation strategy provides a generalized reference to advance versatile practical applications of electroactive microorganisms.

Obtaining electroactive microbes capable of efficient extracellular electron transfer is a large undertaking for the scalability of bio-electrochemical systems. Inevitably, researchers need to pursue the co-modification of multiple genes rather than expecting that modification of a single gene would make a significant contribution to improving extracellular electron transfer rates. Base editing has enabled highly-efficient gene deactivation in model electroactive microbe

The production of the aglycosylated immunoglobulin G (IgG) in Escherichia coli 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, aceF; module 2, gltA and acnA; module 3, serB) and optimization of fermentation conditions, the recombinant E. coli 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 E. coli.

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.

Electroactive microorganisms (EAMs) are ubiquitous in nature and have attracted considerable attention as they can be used for energy recovery and environmental remediation via their extracellular electron transfer (EET) capabilities. Although the EET mechanisms of Shewanella and Geobacter have been rigorously investigated and are well characterized, much less is known about the EET mechanisms of other microorganisms. For EAMs, efficient EET is crucial for the sustainable economic development of bioelectrochemical systems (BESs). Currently, the low efficiency of EET remains a key factor in limiting the development of BESs. In this review, we focus on the EET mechanisms of different microorganisms, (i.e., bacteria, fungi, and archaea). In addition, we describe in detail three engineering strategies for improving the EET ability of EAMs: (1) enhancing transmembrane electron transport via cytochrome protein channels; (2) accelerating electron transport via electron shuttle synthesis and transmission; and (3) promoting the microbe-electrode interface reaction via regulating biofilm formation. At the end of this review, we look to the future, with an emphasis on the cross-disciplinary integration of systems biology and synthetic biology to build high-performance EAM systems.

The production of the aglycosylated immunoglobulin G (IgG) in

Low efficiency in photo-regeneration of redox-active cofactors is a crucial bottleneck in restricting artificial bio-photosynthesis of fuel systems from practical applications. We herein developed novel thiophene-modified doubleshell hollow g-C3N4 nanospheres (ATCN-DSCN) via synergistically enhancing both the charge excitation and separation processes to efficiently photo-regenerate nicotinamide adenine dinucleotide (NADH), which was then utilized as the cofactor by formate dehydrogenase (FDH) to reduce CO2 to form formic acid. The ATCN-DSCN material exhibited outstanding optical and photoelectrical properties, enabling a NADH yield of ∼74%, which was ∼40 times higher than that of the bulk C3N4 (∼1.76%). ATCN-DSCN enabled a turnover frequency (TOF) of 2.950 h−1, which, to the best of our knowledge, is the highest record of TOF for the photo-regeneration of NADH. For the artificial bio-photoreduction of CO2, sustainable conversion of CO2 to formic acid was achieved with a final formic acid concentration of 290.0 μM after 9 hours of light illumination. The excellent optical and photoelectrical properties of the ATCN-DSCN were enabled by the synergistic effect between the specific porous multishell hollow structure and thiophene ring incorporation, which endowed ATCN-DSCN enhanced light absorption and improved charge separation with boosted photocatalytic regeneration of NADH.

NAD(P)H are the cofactors of the cytochrome P450 monooxygenases for the catalytic hydroxylation of steroids, which is notoriously challenging for traditional synthetic chemistry due to the requirement of specificity and chirality. However, an undersupply of NAD(P)H is a crucial bottleneck in restricting the conversion rate of P450-catalyzed steroid hydroxylation. To address the problem of cofactor undersupply and enhance the biotransformation from dehydroepiandrosterone (DHEA) to 7α-OH-DHEA, an electricity-driven NAD(P)H regeneration shortcut was incorporated into recombinant Saccharomyces cerevisiae harbouring 7α-hydroxylase (CYP7B1) to construct a P450 bioelectrocatalytic system (BES). The BES could collaboratively utilize electrons derived from both electrodes and the oxidation of carbon sources, enabling intracellular NAD(P)H regeneration via the concomitant electron shuttle (i.e., neutral red (NR))-mediated extracellular electron transfer (EET) pathway and the oxidation of glucose to facilitate the P450 enzyme-catalysed hydroxylation of steroids. Thus, the 7α-OH-DHEA yield reached 288.6 ± 7.8 mg L−1 in the BES, ∼2.4-fold its counterpart in the absence of EET (122.1 ± 3.7 mg L−1). For the first time, a NR-mediated BES was exhibited to be an efficient strategy to enhance steroid hydroxylation in engineered yeast, being promising for steroid conversion in pharmaceutical engineering.

Menaquinone-7 (MK-7), a valuable vitamin K2, plays an important role in the prevention of osteoporosis and cardiovascular calcification. We chose B. subtilis 168 as the chassis for the modular metabolic engineering design to promote the biosynthesis of MK-7. The biosynthetic pathway of MK-7 was categorized into four modules, namely, the MK-7 pathway (Module I), the shikimate (SA) pathway (Module II), the methylerythritol phosphate (MEP) pathway (Module III), and the glycerol metabolism pathway (Module IV). Overexpression of menA (Module I) resulted in 6.6 ± 0.1 mg/L of MK-7 after 120 h fermentation, which was 2.1-fold that of the starting strain BS168NU (3.1 ± 0.2 mg/L). Overexpression of aroA, aroD, and aroE (Module II) had a negative effect on the synthesis of MK-7. Simultaneous overexpression of dxs, dxr, yacM, and yacN (Module III) enabled the yield of MK-7 to 12.0 ± 0.1 mg/L. Moreover, overexpression of glpD (Module IV) resulted in an increase of the yield of MK-7 to 13.7 ± 0.2 mg/L. Furthermore, del...

The slow rate of extracellular electron transfer (EET) of electroactive microorganisms remains a primary bottleneck that restricts the practical applications of bioelectrochemical systems. Intracellular NAD(H/+) (i.e., the total level of NADH and NAD+) is a crucial source of the intracellular electron pool from which intracellular electrons are transferred to extracellular electron acceptors via EET pathways. However, how the total level of intracellular NAD(H/+) impacts the EET rate in Shewanella oneidensis has not been established. Here, we use a modular synthetic biology strategy to redirect metabolic flux towards NAD+ biosynthesis via three modules: de novo, salvage, and universal biosynthesis modules in S. oneidensis MR-1. The results demonstrate that an increase in intracellular NAD(H/+) results in the transfer of more electrons from the increased oxidation of the electron donor to the EET pathways of S. oneidensis, thereby enhancing intracellular electron flux and the EET rate., A bottleneck for the application of bioelectrochemical systems is the slow rate of extracellular electron transfer. Here the authors use a synthetic biology approach to redirect metabolic flux to NAD+ biosynthesis, which enhances the intracellular electron flux and the extracellular electron transfer rate.

Extracellular electron transfer (EET) in Shewanella oneidensis MR-1, which is one of the most well-studied exoelectrogens, underlies many microbial electrocatalysis processes, including microbial fuel cells, microbial electrolysis cells, and microbial electrosynthesis. However, regulating the efficiency of EET remains challenging due to the lack of efficient genome regulation tools that regulate gene expression levels in S. oneidensis. Here, we systematically established a transcriptional regulation technology, i.e., clustered regularly interspaced short palindromic repeats interference (CRISPRi), in S. oneidensis MR-1 using green fluorescent protein (GFP) as a reporter. We used this CRISPRi technology to repress the expression levels of target genes, individually and in combination, in the EET pathways (e.g., the MtrCAB pathway and genes affecting the formation of electroactive biofilms in S. oneidensis), which in turn enabled the efficient regulation of EET efficiency. We then established a translational regulation technology, i.e., Hfq-dependent small regulatory RNA (sRNA), in S. oneidensis by repressing the GFP reporter and mtrA, which is a critical gene in the EET pathways in S. oneidensis. To achieve coordinated transcriptional and translational regulation at the genomic level, the CRISPRi and Hfq-dependent sRNA systems were incorporated into a single plasmid harbored in a recombinant S. oneidensis strain, which enabled an even higher efficiency of mtrA gene repression in the EET pathways than that achieved by the CRISPRi and Hfq-dependent sRNA system alone, as exhibited by the reduced electricity output. Overall, we developed a combined CRISPRi-sRNA method that enabled the synergistic transcriptional and translational regulation of target genes in S. oneidensis. This technology involving CRISPRi-sRNA transcriptional-translational regulation of gene expression at the genomic level could be applied to other microorganisms.

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 is a platform microorganism for understanding extracellular electron transfer (EET) with a fully sequenced and annotated genome. In comparison to other model microorganisms such as Escherichia coli, the available plasmid parts (such as promoters and replicons) are not sufficient to conveniently and quickly fine-tune the expression of multiple genes in S. oneidensis MR-1. Here, we constructed and characterized a plasmid toolkit that contains a set of expression vectors with a combination of promoters, replicons, antibiotic resistance genes, and an RK2 origin of transfer (oriT) cassette, in which each element can be easily changed by fixed restriction enzyme sites. The expression cassette is also compatible with BioBrick synthetic biology standards. Using green fluorescent protein (GFP) as a reporter, we tested and quantified the strength of promoters. The copy number of different replicons was also measured by real-time quantitative PCR. We further transformed two compatible plasmids with different antibiotic resistance genes into the recombinant S. oneidensis MR-1, enabling control over the expression of two different fluorescent proteins. This plasmid toolkit was further used for overexpression of the MtrCAB porin-c-type cytochrome complex in the S. oneidensis ΔmtrA strain. Tungsten trioxide (WO3) reduction and microbial fuel cell (MFC) assays revealed that the EET efficiency was improved most significantly when MtrCAB was expressed at a moderate level, thus demonstrating the utility of the plasmid toolkit in the EET regulation in S. oneidensis. The plasmid toolkit developed in this study is useful for rapid and convenient fine-tuning of gene expression and enhances the ability to genetically manipulate S. oneidensis MR-1.

Additional file 1: Table S1. The sequences of sfp and dCas9.

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.

Efficient extracellular electron transfer (EET) of exoelectrogens is essentially for practical applications of versatile bioelectrochemical systems. Intracellular electrons flow from NADH to extracellular electron acceptors via EET pathways. However, it was yet established how the manipulation of intracellular NADH impacted the EET efficiency. Strengthening NADH regeneration from NAD+, as a feasible approach for cofactor engineering, has been used in regulating the intracellular NADH pool and the redox state (NADH/NAD+ ratio) of cells. Herein, we first adopted a modular metabolic engineering strategy to engineer and drive the metabolic flux toward the enhancement of intracellular NADH regeneration. We systematically studied 16 genes related to the NAD+-dependent oxidation reactions for strengthening NADH regeneration in the four metabolic modules of S. oneidensis MR-1, i.e., glycolysis, C1 metabolism, pyruvate fermentation, and tricarboxylic acid cycle. Among them, three endogenous genes mostly responsibl...

The microbial fuel cell (MFC) is a promising technology for energy harvesting from biomass; however, previously reported MFCs with wild-type or biologically modified exoelectrogenic bacteria such as Shewanella oneidensis have often exhibited poor performance in generating electricity from sugars. Herein, a synthetic fermenter-exoelectrogen (Escherichia coli-S. oneidensis) microbial consortium was developed to expand the spectrum of carbon sources for MFC through establishing a highly electroactive anodic biofilm by rationally tuning its microbial community profile to favor efficient electron transfer. Specifically, a synthetic riboflavin pathway from Bacillus subtilis was incorporated into E. coli to overproduce flavins to facilitate flavin-mediated electron transfer, and a highly hydrophobic S. oneidensis strain CP2-1-S1 was adopted as the exoelectrogen to increase its adhesion to the carbon electrode. The highly hydrophobic interactions between S. oneidensis and the anode along with the overproduced fla...

It is hard to get the pressure of high speed water column on the inner barrel through the gun water-projectile test. In order to optimize the design, the calculation model of the pressure in the body tube is established. Two dimensional numerical simulation was carried out for gun water-projectile test's high speed water column in the body tube through using Fluent software, the distribution of turbulence was described by using the k-e model and the gas-liquid interface of the inside body tube was tracked through two-dimensional VOF model. It was got that simulation of the evolution of the gas - liquid interface image, as well as the pressure distribution at different time in the tube. From the simulation results, it can be seen that the high speed water column will have great pressure on the pipe wall, and the results will be of some guiding significance for the improvement of the gun water-projectile test.