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.
•Extracellular electron transfer mechanisms are summarized.•Extracellular electron transport pathways of microorganisms are summarized.•Engineering strategies for improving EET capabilities are reviewed.•New insights on further EET research are presented.
Abstract
To construct a superior microbial cell factory for chemical synthesis, a major challenge is to fully exploit cellular potential by identifying and engineering beneficial gene targets in ...sophisticated metabolic networks. Here, we take advantage of CRISPR interference (CRISPRi) and omics analyses to systematically identify beneficial genes that can be engineered to promote free fatty acids (FFAs) production in
Escherichia coli
. CRISPRi-mediated genetic perturbation enables the identification of 30 beneficial genes from 108 targets related to FFA metabolism. Then, omics analyses of the FFAs-overproducing strains and a control strain enable the identification of another 26 beneficial genes that are seemingly irrelevant to FFA metabolism. Combinatorial perturbation of four beneficial genes involving cellular stress responses results in a recombinant strain
ihfA
L−
-
aidB
+
-
ryfA
M−
-
gadA
H−
, producing 30.0 g L
−1
FFAs in fed-batch fermentation, the maximum titer in
E. coli
reported to date. Our findings are of help in rewiring cellular metabolism and interwoven intracellular processes to facilitate high-titer production of biochemicals.
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.
Surfactin is a cyclic lipopeptide that is of great industrial use owing to its extraordinary surfactant power and antimicrobial, antiviral, and antitumor activities. Surfactin is synthesized by a ...condensation reaction in microbes, which uses fatty acids and four kinds of amino acids (L-glutamate, L-aspartate, L-leucine and L-valine) as precursors. Surfactin biosynthesis could be improved by increasing the supply of fatty acids; however, the effect of the regulation of amino acid metabolism on surfactin production was not yet clear.
In this study, we aimed to improve surfactin production in B. subtilis by repressing the genes on the branch metabolic pathways of amino acid biosynthesis using CRISPRi technology. First, 20 genes were inhibited individually, resulting in 2.5- to 627-fold decreases in transcriptional level as determined by RT-qPCR. Among the 20 recombinant strains, 16 strains obtained higher surfactin titres than that produced by the parent BS168NU-Sd strain (the surfactin production of BS168NU-Sd with only dCas9 but no sgRNA expression was 0.17 g/L). In particular, the strains in which the yrpC, racE or murC genes were inhibited individually produced 0.54, 0.41, or 0.42 g/L surfactin, respectively. All three genes are related to the metabolism of L-glutamate, whose acylation is the first step in the surfactin condensation reaction. Furthermore, these three genes were repressed in combination, and the strain with co-inhibition of yrpC and racE produced 0.75 g/L surfactin, which was 4.69-fold higher than that of the parent strain. In addition, the inhibition of bkdAA and bkdAB, which are related to the metabolism of L-leucine and L-valine, not only improved surfactin production but also increased the proportion of the C
isoform.
This study, to the best of our knowledge for the first time, systematically probed the regulatory effect of increasing the supply of amino acids on surfactin production. It provided an effective strategy and a new perspective for systematic studies on surfactin and other amino acid-derived chemicals.
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
, the ...available plasmid parts (such as promoters and replicons) are not sufficient to conveniently and quickly fine-tune the expression of multiple genes in
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 (
) 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
MR-1, enabling control over the expression of two different fluorescent proteins. This plasmid toolkit was further used for overexpression of the MtrCAB porin-
-type cytochrome complex in the
Δ
strain. Tungsten trioxide (WO
) 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
. 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
MR-1.
Genomic variants libraries are conducive to obtain dominant strains with desirable phenotypic traits. The non-homologous end joining (NHEJ), which enables foreign DNA fragments to be randomly ...integrated into different chromosomal sites, shows prominent capability in genomic libraries construction. In this study, we established an efficient NHEJ-mediated genomic library technology in Yarrowia lipolytica through regulation of NHEJ repair process, employment of defective Ura marker and optimization of iterative transformations, which enhanced genes integration efficiency by 4.67, 22.74 and 1.87 times, respectively. We further applied this technology to create high lycopene producing strains by multi-integration of heterologous genes of CrtE, CrtB and CrtI, with 23.8 times higher production than rDNA integration through homologous recombination (HR). The NHEJ-mediated genomic library technology also achieved random and scattered integration of loxP and vox sites, with the copy number up to 65 and 53, respectively, creating potential for further application of recombinase mediated genome rearrangement in Y. lipolytica. This work provides a high-efficient NHEJ-mediated genomic library technology, which enables random and scattered genomic integration of multiple heterologous fragments and rapid generation of diverse strains with superior phenotypes within 96 h. This novel technology also lays an excellent foundation for the development of other genetic technologies in Y. lipolytica.
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 responsible for increasing NADH regeneration were identified, namely gapA2 encoding a NAD+-dependent glyceraldehyde-3-phosphate dehydrogenase in the glycolysis module, mdh encoding a NAD+-dependent malate dehydrogenase in the TCA cycle, and pflB encoding a pyruvate-formate lyase that converted pyruvate to formate in the pyruvate fermentation module. An exogenous gene fdh* from Candida boidinii encoding a NAD+-dependent formate dehydrogenase to increase NADH regeneration in the pyruvate fermentation module was further identified. Upon assembling these four genes in S. oneidensis MR-1, ∼4.3-fold increase in NADH/NAD+ ratio, and ∼1.2-fold increase in intracellular NADH pool were obtained under anaerobic conditions without discharge, which elicited ∼3.0-fold increase in the maximum power output in microbial fuel cells, from 26.2 ± 2.8 (wild-type) to 105.8 ± 4.1 mW/m2 (recombinant S. oneidensis), suggesting a boost in the EET efficiency. This modular engineering method in controlling the intracellular reducing equivalents would be a general approach in tuning the EET efficiency of exoelectrogens.
Microbial fuel cells (MFCs) are eco-friendly bio-electrochemical reactors that use exoelectrogens as biocatalyst for electricity harvest from organic biomass, which could also be used as biosensors ...for long-term environmental monitoring. Glucose and xylose, as the primary ingredients from cellulose hydrolyzates, is an appealing substrate for MFC. Nevertheless, neither xylose nor glucose can be utilized as carbon source by well-studied exoelectrogens such as
. In this study, to harvest the electricity by rapidly harnessing xylose and glucose from corn stalk hydrolysate, we herein firstly designed glucose and xylose co-fed engineered
microbial consortium, in which
as the fermenter converted glucose and xylose into lactate to feed the exoelectrogens (
). To produce more lactate in
, we eliminated the ethanol and acetate pathway via deleting
(phosphotransacetylase gene) and
(alcohol dehydrogenase gene) and further constructed a synthesis and delivery system through expressing
(lactate dehydrogenase gene) and
(lactate transporter gene). To facilitate extracellular electron transfer (EET) of
, a biosynthetic flavins pathway from
was expressed in a highly hydrophobic
CP-S1, which not only improved direct-contacted EET via enhancing
adhesion to the carbon electrode but also accelerated the flavins-mediated EET via increasing flavins synthesis. Furthermore, we optimized the ratio of glucose and xylose concentration to provide a stable carbon source supply in MFCs for higher power density. The glucose and xylose co-fed MFC inoculated with the recombinant consortium generated a maximum power density of 104.7 ± 10.0 mW/m
, which was 7.2-folds higher than that of the wild-type consortium (12.7 ± 8.0 mW/m
). Lastly, we used this synthetic microbial consortium in the corn straw hydrolyzates-fed MFC, obtaining a power density 23.5 ± 6.0 mW/m
.
Non-homologous end joining (NHEJ)-mediated integration is effective in generating random mutagenesis to identify beneficial gene targets in the whole genome, which can significantly promote the ...performance of the strains. Here, a novel target leading to higher protein synthesis was identified by NHEJ-mediated integration that seriously improved fatty alcohols biosynthesis in
. One batch of strains transformed with fatty acyl-CoA reductase gene (
) showed significant differences (up to 70.53-fold) in fatty alcohol production. Whole-genome sequencing of the high-yield strain demonstrated that a new target YALI0_A00913g ("A1 gene") was disrupted by NHEJ-mediated integration of partial carrier DNA, and reverse engineering of the A1 gene disruption (YlΔA1-FAR) recovered the fatty alcohol overproduction phenotype. Transcriptome analysis of YlΔA1-FAR strain revealed A1 disruption led to strengthened protein synthesis process that was confirmed by
gene expression, which may account for enhanced cell viability and improved biosynthesis of fatty alcohols. This study identified a novel target that facilitated synthesis capacity and provided new insights into unlocking biosynthetic potential for future genetic engineering in
.
Bio-electrochemical systems are based on extracellular electron transfer (EET), whose efficiency relates to the expression level of numerous genes. However, the lack of multi-functional tools for ...gene activation and repression hampers the enhancement of EET in electroactive microorganisms (EAMs). We thus develop a type I-F CRISPR/PaeCascade-RpoD-mediated activation and inhibition regulation (CRISPR-PAIR) platform in the model EAM, Shewanella oneidensis MR-1. Gene activation is achieved (3.8-fold) through fusing activator RpoD (σ70) to Cas7 when targeting the prioritized loci upstream of the transcription start site. Gene inhibition almost has no position preference when targeting the open reading frame, which makes the design of crRNAs easy and flexible. Then CRISPR-PAIR platform is applied to up-/down-regulate the expression of six endogenous genes, resulting in the improved EET efficiency. Moreover, simultaneous gene activation and inhibition are achieved in S. oneidensis MR-1. CRISPR-PAIR platform offers a programmable methodology for dual regulation, facilitating in-depth EET studies in Shewanella spp.
Display omitted
•CRISPR-PAIR platform enables both gene activation and inhibition in Shewanella oneidensis•An efficient type I-F CRISPR-Cas tool is developed for S. oneidensis•Transcription regulation of endogenous genes enhances extracellular electron transfer (EET)
Biochemistry; Biochemical engineering; Metabolic engineering
PDF
