Synthetic biology on acetogenic bacteria for highly efficient conversion of c1 gases to biochemicals

© 2020 by the authors. Licensee MDPI, Basel, Switzerland. Synthesis gas, which is mainly produced from fossil fuels or biomass gasification, consists of C1 gases such as carbon monoxide, carbon dioxide, and methane as well as hydrogen. Acetogenic bacteria (acetogens) have emerged as an alternative solution to recycle C1 gases by converting them into value-added biochemicals using the Wood-Ljungdahl pathway. Despite the advantage of utilizing acetogens as biocatalysts, it is difficult to develop industrial-scale bioprocesses because of their slow growth rates and low productivities. To solve these problems, conventional approaches to metabolic engineering have been applied; however, there are several limitations owing to the lack of required genetic bioparts for regulating their metabolic pathways. Recently, synthetic biology based on genetic parts, modules, and circuit design has been actively exploited to overcome the limitations in acetogen engineering. This review covers synthetic biology applications to design and build industrial platform acetogens

Elucidation of Akkermansia muciniphila Probiotic Traits Driven by Mucin Depletion

Akkermansia muciniphila is widely considered a next-generation beneficial microbe. This bacterium resides in the mucus layer of its host and regulates intestinal homeostasis and intestinal barrier integrity by affecting host signaling pathways. However, it remains unknown how the expression of genes encoding extracellular proteins is regulated in response to dynamic mucosal environments. In this study, we elucidated the effect of mucin on the gene expression and probiotic traits of A. muciniphila. Transcriptome analysis showed that the genes encoding most mucin-degrading enzymes were significantly upregulated in the presence of mucin. By contrast, most genes involved in glycolysis and energy metabolic pathways were upregulated under mucin-depleted conditions. Interestingly, the absence of mucin resulted in the upregulation of 79 genes encoding secreted protein candidates, including Amuc-1100 as well as members of major protein secretion systems. These transcript level changes were consistent with the fact that administration of A. muciniphila grown under mucin-depleted conditions to high-fat diet-induced diabetic mice reduced obesity and improved intestinal barrier integrity more efficiently than administration of A. muciniphila grown under mucin-containing conditions. In conclusion, mucin content in the growth medium plays a critical role in the improvement by A. muciniphila of high-fat diet-induced obesity, intestinal inflammation, and compromised intestinal barrier integrity related to a decrease in goblet cell density. Our findings suggest the depletion of animal-derived mucin in growth medium as a novel principle for the development of A. muciniphila for human therapeutics

Applications of CRISPR/Cas System to Bacterial Metabolic Engineering

The clustered regularly interspaced short palindromic repeats/CRISPR-associated (CRISPR/Cas) adaptive immune system has been extensively used for gene editing, including gene deletion, insertion, and replacement in bacterial and eukaryotic cells owing to its simple, rapid, and efficient activities in unprecedented resolution. Furthermore, the CRISPR interference (CRISPRi) system including deactivated Cas9 (dCas9) with inactivated endonuclease activity has been further investigated for regulation of the target gene transiently or constitutively, avoiding cell death by disruption of genome. This review discusses the applications of CRISPR/Cas for genome editing in various bacterial systems and their applications. In particular, CRISPR technology has been used for the production of metabolites of high industrial significance, including biochemical, biofuel, and pharmaceutical products/precursors in bacteria. Here, we focus on methods to increase the productivity and yield/titer scan by controlling metabolic flux through individual or combinatorial use of CRISPR/Cas and CRISPRi systems with introduction of synthetic pathway in industrially common bacteria including Escherichia coli. Further, we discuss additional useful applications of the CRISPR/Cas system, including its use in functional genomics

Systems biology of competency in Vibrio natriegens is revealed by applying novel data analytics to the transcriptome

Summary: Vibrio natriegens regulates natural competence through the TfoX and QstR transcription factors, which are involved in external DNA capture and transport. However, the extensive genetic and transcriptional regulatory basis for competency remains unknown. We used a machine-learning approach to decompose Vibrio natriegens’s transcriptome into 45 groups of independently modulated sets of genes (iModulons). Our findings show that competency is associated with the repression of two housekeeping iModulons (iron metabolism and translation) and the activation of six iModulons; including TfoX and QstR, a novel iModulon of unknown function, and three housekeeping iModulons (representing motility, polycations, and reactive oxygen species [ROS] responses). Phenotypic screening of 83 gene deletion strains demonstrates that loss of iModulon function reduces or eliminates competency. This database-iModulon-discovery cycle unveils the transcriptomic basis for competency and its relationship to housekeeping functions. These results provide the genetic basis for systems biology of competency in this organism

Analysis of the core genome and pan-genome of autotrophic acetogenic bacteria

Acetogens are obligate anaerobic bacteria capable of reducing carbon dioxide (CO2) to multicarbon compounds coupled to the oxidation of inorganic substrates, such as hydrogen (H2) or carbon monoxide (CO), via the Wood-Ljungdahl pathway. Owing to the metabolic capability of CO2 fixation, much attention has been focused on understanding the unique pathways associated with acetogens, particularly their metabolic coupling of CO2 fixation to energy conservation. Most known acetogens are phylogenetically and metabolically diverse bacteria present in 23 different bacterial genera. With the increased volume of available genome information, acetogenic bacterial genomes can be analyzed by comparative genome analysis. Even with the genetic diversity that exists among acetogens, the Wood-Ljungdahl pathway, a central metabolic pathway, and cofactor biosynthetic pathways are highly conserved for autotrophic growth. Additionally, comparative genome analysis revealed that most genes in the acetogen-specific core genome were associated with the Wood-Ljungdahl pathway. The conserved enzymes and those predicted as missing can provide insight into biological differences between acetogens and allow for the discovery of promising candidates for industrial applications

Genome-scale analysis of syngas fermenting acetogenic bacteria reveals the translational regulation for its autotrophic growth

Abstract Background Acetogenic bacteria constitute promising biocatalysts for the conversion of CO2/H2 or synthesis gas (H2/CO/CO2) into biofuels and value-added biochemicals. These microorganisms are naturally capable of autotrophic growth via unique acetogenesis metabolism. Despite their biosynthetic potential for commercial applications, a systemic understanding of the transcriptional and translational regulation of the acetogenesis metabolism remains unclear. Results By integrating genome-scale transcriptomic and translatomic data, we explored the regulatory logic of the acetogenesis to convert CO2 into biomass and metabolites in Eubacterium limosum. The results indicate that majority of genes associated with autotrophic growth including the Wood-Ljungdahl pathway, the reduction of electron carriers, the energy conservation system, and gluconeogenesis were transcriptionally upregulated. The translation efficiency of genes in cellular respiration and electron bifurcation was also highly enhanced. In contrast, the transcriptionally abundant genes involved in the carbonyl branch of the Wood-Ljungdahl pathway, as well as the ion-translocating complex and ATP synthase complex in the energy conservation system, showed decreased translation efficiency. The translation efficiencies of genes were regulated by 5′UTR secondary structure under the autotrophic growth condition. Conclusions The results illustrated that the acetogenic bacteria reallocate protein synthesis, focusing more on the translation of genes for the generation of reduced electron carriers via electron bifurcation, rather than on those for carbon metabolism under autotrophic growth

Functional cooperation of the glycine synthase-reductase and Wood???Ljungdahl pathways for autotrophic growth of Clostridium drakei

Among CO2-fixing metabolic pathways in nature, the linear Wood-Ljungdahl pathway (WLP) in phylogenetically diverse acetate-forming acetogens comprises the most energetically efficient pathway, requires the least number of reactions, and converts CO2 to formate and then into acetyl-CoA. Despite two genes encoding glycine synthase being well-conserved in WLP gene clusters, the functional role of glycine synthase under autotrophic growth conditions has remained uncertain. Here, using the reconstructed genomescale metabolic model iSL771 based on the completed genome sequence, transcriptomics, C-13 isotope-based metabolite-tracing experiments, biochemical assays, and heterologous expression of the pathway in another acetogen, we discovered that the WLP and the glycine synthase pathway are functionally interconnected to fix CO2, subsequently converting CO2 into acetyl-CoA, acetyl-phosphate, and serine. Moreover, the functional cooperation of the pathways enhances CO2 consumption and cellular growth rates via bypassing reducing power required reactions for cellular metabolism during autotrophic growth of acetogens