scholarly journals Design of a programmable biosensor-CRISPRi genetic circuits for dynamic and autonomous dual-control of metabolic flux in Bacillus subtilis

2019 ◽  
Vol 48 (2) ◽  
pp. 996-1009 ◽  
Author(s):  
Yaokang Wu ◽  
Taichi Chen ◽  
Yanfeng Liu ◽  
Rongzhen Tian ◽  
Xueqin Lv ◽  
...  
Keyword(s):  
Bacillus Subtilis ◽  
Metabolic Pathways ◽  
Metabolic Flux ◽  
Target Genes ◽  
Fine Tuning ◽  
Dual Control ◽  
Genetic Circuits ◽  
Dynamic Regulation ◽  
Batch Bioreactor ◽  
Acetoin Production

Abstract Dynamic regulation is an effective strategy for fine-tuning metabolic pathways in order to maximize target product synthesis. However, achieving dynamic and autonomous up- and down-regulation of the metabolic modules of interest simultaneously, still remains a great challenge. In this work, we created an autonomous dual-control (ADC) system, by combining CRISPRi-based NOT gates with novel biosensors of a key metabolite in the pathway of interest. By sensing the levels of the intermediate glucosamine-6-phosphate (GlcN6P) and self-adjusting the expression levels of the target genes accordingly with the GlcN6P biosensor and ADC system enabled feedback circuits, the metabolic flux towards the production of the high value nutraceutical N-acetylglucosamine (GlcNAc) could be balanced and optimized in Bacillus subtilis. As a result, the GlcNAc titer in a 15-l fed-batch bioreactor increased from 59.9 g/l to 97.1 g/l with acetoin production and 81.7 g/l to 131.6 g/l without acetoin production, indicating the robustness and stability of the synthetic circuits in a large bioreactor system. Remarkably, this self-regulatory methodology does not require any external level of control such as the use of inducer molecules or switching fermentation/environmental conditions. Moreover, the proposed programmable genetic circuits may be expanded to engineer other microbial cells and metabolic pathways.

CRISPRi-Guided Multiplexed Fine-Tuning of Metabolic Flux for Enhanced Lacto-N-neotetraose Production in Bacillus subtilis

2020 ◽  
Vol 68 (8) ◽  
pp. 2477-2484 ◽  
Author(s):  
Xiaomin Dong ◽  
Nan Li ◽  
Zhenmin Liu ◽  
Xueqin Lv ◽  
Yu Shen ◽  
...  
Keyword(s):  
Bacillus Subtilis ◽  
Metabolic Flux ◽  
Fine Tuning

scholarly journals Optimising PHBV biopolymer production in haloarchaea via CRISPRi-mediated redirection of carbon flux

2021 ◽  
Vol 4 (1) ◽  
Author(s):  
Lin Lin ◽  
Junyu Chen ◽  
Ruchira Mitra ◽  
Quanxiu Gao ◽  
Feiyue Cheng ◽  
...  
Keyword(s):  
Gene Expression ◽  
Metabolic Pathways ◽  
Carbon Flux ◽  
Metabolic Flux ◽  
Citrate Synthase ◽  
Fine Tuning ◽  
Type I ◽  
Haloferax Mediterranei ◽  
Target Region ◽  
Fermentation Period

AbstractThe haloarchaeon Haloferax mediterranei is a potential strain for poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) production, yet the production yield and cost are the major obstacles hindering the use of this archaeal strain. Leveraging the endogenous type I-B CRISPR-Cas system in H. mediterranei, we develop a CRISPR-based interference (CRISPRi) approach that allows to regulate the metabolic pathways related to PHBV synthesis, thereby enhancing PHBV production. Our CRISPRi approach can downregulate the gene expression in a range of 25% to 98% depending upon the target region. Importantly, plasmid-mediated CRISPRi downregulation on the citrate synthase genes (citZ and gltA) improves the PHBV accumulation by 76.4% (from 1.78 to 3.14 g/L). When crRNA cassette integrated into chromosome, this further shortens the PHBV fermentation period and enhances PHA productivity by 165%. Our transcriptome analysis shows that repression of citrate synthase genes redirects metabolic flux from the central metabolic pathways to PHBV synthesis pathway. These findings demonstrate that the CRISPRi-based gene regulation is a transformative toolkit for fine-tuning the endogenous metabolic pathways in the archaeal system, which can be applied to not only the biopolymer production but also many other applications.

scholarly journals Co-production of acetoin and succinic acid by metabolically engineered Enterobacter cloacae

2021 ◽  
Vol 14 (1) ◽  
Author(s):  
Hsiang-Yen Su ◽  
Hua-Ying Li ◽  
Cai-Yun Xie ◽  
Qiang Fei ◽  
Ke-Ke Cheng
Keyword(s):  
Succinic Acid ◽  
Acid Production ◽  
Metabolic Flux ◽  
Lactic Acid Production ◽  
Enterobacter Cloacae ◽  
Culture Conditions ◽  
Single Step ◽  
Fine Tuning ◽  
Renewable Chemicals ◽  
Acetoin Production

Abstract Background Renewable chemicals have attracted attention due to increasing interest in environmental concerns and resource utilization. Biobased production of industrial compounds from nonfood biomass has become increasingly important as a sustainable replacement for traditional petroleum-based production processes depending on fossil resources. Therefore, we engineered an Enterobacter cloacae budC and ldhA double-deletion strain (namely, EC∆budC∆ldhA) to redirect carbon fluxes and optimized the culture conditions to co-produce succinic acid and acetoin. Results In this work, E. cloacae was metabolically engineered to enhance its combined succinic acid and acetoin production during fermentation. Strain EC∆budC∆ldhA was constructed by deleting 2,3-butanediol dehydrogenase (budC), which is involved in 2,3-butanediol production, and lactate dehydrogenase (ldhA), which is involved in lactic acid production, from the E. cloacae genome. After redirecting and fine-tuning the E. cloacae metabolic flux, succinic acid and acetoin production was enhanced, and the combined production titers of acetoin and succinic acid from glucose were 17.75 and 2.75 g L−1, respectively. Moreover, to further improve acetoin and succinic acid production, glucose and NaHCO3 modes and times of feeding were optimized during fermentation of the EC∆budC∆ldhA strain. The maximum titers of acetoin and succinic acid were 39.5 and 20.3 g L−1 at 72 h, respectively. Conclusions The engineered strain EC∆budC∆ldhA is useful for the co-production of acetoin and succinic acid and for reducing microbial fermentation costs by combining processes into a single step.

scholarly journals Development of a growth coupled dynamic regulation network balancing malonyl-CoA node to enhance (2S)-naringenin synthesis in E. coli

2020 ◽  
Author(s):  
Shenghu Zhou ◽  
Shuo-Fu Yuan ◽  
Priya H Nair ◽  
Hal S Alper ◽  
Yu Deng ◽  
...  
Keyword(s):  
Fatty Acid Synthase ◽  
Metabolic Flux ◽  
High Efficiency ◽  
Acyl Carrier Protein ◽  
Single Cells ◽  
Fine Tuning ◽  
Coumaric Acid ◽  
Dynamic Regulation ◽  
Trade Offs ◽  
Malonyl Coa

AbstractGenerally, high- and low-performance nongenetic variants and young and aged cells co-existed in culture at all growth phases. In this regard, individually and dynamically regulating the metabolic flux of single cells based on their cellular state is highly useful for improving the performance of populations. However, balancing the trade-offs between biomass formation and compound over-production requires sophisticated dynamic regulation networks (DRNs) which can be challenging. Here, we developed a growth coupled NCOMB (Naringenin-Coumaric acid-Malonyl-CoA-Balanced) DRN with systematic optimization of (2S)-naringenin and p-coumaric acid-responsive regulation pathways for real-time control of intracellular supply of malonyl-CoA. In doing so, the acyl carrier protein was used as a new critical node for fine-tuning malonyl-CoA consumption instead of fatty acid synthase. Following directed evolution of the NCOMB DRN, we obtained a strain with cumulative 8.4-fold improvement in (2S)-naringenin production with balanced cell growth, demonstrating the high efficiency of this system for improving pathway production.

Multilayer Genetic Circuits for Dynamic Regulation of Metabolic Pathways

2021 ◽  
Author(s):  
Shixiu Cui ◽  
Xueqin Lv ◽  
Xianhao Xu ◽  
Taichi Chen ◽  
Hongzhi Zhang ◽  
...  
Keyword(s):  
Metabolic Pathways ◽  
Genetic Circuits ◽  
Dynamic Regulation

scholarly journals Data-Driven Prediction of CRISPR-Based Transcription Regulation for Programmable Control of Metabolic Flux

2017 ◽  
Author(s):  
Jiayuan Sheng ◽  
Weihua Guo ◽  
Christine Ash ◽  
Brendan Freitas ◽  
Mitchell Paoletti ◽  
...  
Keyword(s):  
Metabolic Engineering ◽  
Metabolic Flux ◽  
Target Genes ◽  
Product Yield ◽  
Fine Tuning ◽  
Data Driven ◽  
Gene Expressions ◽  
Guide Rnas ◽  
Directional Control ◽  
Transcription Regulators

AbstractMultiplex and multi-directional control of metabolic pathways is crucial for metabolic engineering to improve product yield of fuels, chemicals, and pharmaceuticals. To achieve this goal, artificial transcriptional regulators such as CRISPR-based transcription regulators have been developed to specifically activate or repress genes of interest. Here, we found that by deploying guide RNAs to target on DNA sites at different locations of genetic cassettes, we could use just one synthetic CRISPR-based transcriptional regulator to simultaneously activate and repress gene expressions. By using the pairwise datasets of guide RNAs and gene expressions, we developed a data-driven predictive model to rationally design this system for fine-tuning expression of target genes. We demonstrated that this system could achieve programmable control of metabolic fluxes when using yeast to produce versatile chemicals. We anticipate that this master CRISPR-based transcription regulator will be a valuable addition to the synthetic biology toolkit for metabolic engineering, speeding up the “design-build-test” cycle in industrial biomanufacturing as well as generating new biological insights on the fates of eukaryotic cells.

scholarly journals Increased Fitness and Alteration of Metabolic Pathways during Bacillus subtilis Evolution in the Laboratory

2011 ◽  
Vol 77 (12) ◽  
pp. 4105-4118 ◽  
Author(s):  
Heather Maughan ◽  
Wayne L. Nicholson
Keyword(s):  
Bacillus Subtilis ◽  
Metabolic Pathways ◽  
High Performance ◽  
Colony Morphology ◽  
Flagellar Motility ◽  
Content Type ◽  
Batch Cultures ◽  
Fitness Advantage ◽  
Acetoin Production ◽  
Small Colony

ABSTRACTFive batch cultures ofBacillus subtiliswere subjected to evolution in the laboratory for 6,000 generations under conditions repressing sporulation in complex liquid medium containing glucose. Between generations 1,000 and 2,000, variants with a distinct small-colony morphology arose and swept through four of the five populations that had been previously noted for their loss of sporulation (H. Maughan et al., Genetics 177:937-948, 2007). To better understand the nature of adaptation in these variants, individual strains were isolated from one population before (WN715) and after (WN716) the sweep. In addition to colony morphology, strains WN715 and WN716 differed in their motility, aerotaxis, and cell morphology. Competition experiments showed that strain WN716 had evolved a distinct fitness advantage over the ancestral strain and strain WN715 during growth and the transition to the postexponential growth phase, which was more pronounced when WN715 was present in the coculture. Microarray analyses revealed candidate genes in which mutations may have produced some of the observed phenotypes. For example, loss of motility in WN716 was accompanied by decreased transcription of all flagellar, motility, and chemotaxis genes on the microarray. Transcription ofalsSandalsDwas also lower in strain WN716, and the predicted loss of acetoin production and enhanced acetate production was confirmed by high-performance liquid chromatography (HPLC) analysis. The results suggested that the derived colony morphology of strain WN716 was associated with increased fitness, the alteration of several metabolic pathways, and the loss of a typical postexponential-phase response.

The Synergy between CRISPR and Chemical Engineering

2019 ◽  
Vol 19 (3) ◽  
pp. 147-171
Author(s):  
Cia-Hin Lau ◽  
Chung Tin
Keyword(s):  
Viral Vectors ◽  
Chemical Engineering ◽  
Target Genes ◽  
New Technologies ◽  
Rapid Development ◽  
Genetic Circuits ◽  
Viral Packaging ◽  
Conditional Control ◽  
Logic Operations

Gene therapy and transgenic research have advanced quickly in recent years due to the development of CRISPR technology. The rapid development of CRISPR technology has been largely benefited by chemical engineering. Firstly, chemical or synthetic substance enables spatiotemporal and conditional control of Cas9 or dCas9 activities. It prevents the leaky expression of CRISPR components, as well as minimizes toxicity and off-target effects. Multi-input logic operations and complex genetic circuits can also be implemented via multiplexed and orthogonal regulation of target genes. Secondly, rational chemical modifications to the sgRNA enhance gene editing efficiency and specificity by improving sgRNA stability and binding affinity to on-target genomic loci, and hence reducing off-target mismatches and systemic immunogenicity. Chemically-modified Cas9 mRNA is also more active and less immunogenic than the native mRNA. Thirdly, nonviral vehicles can circumvent the challenges associated with viral packaging and production through the delivery of Cas9-sgRNA ribonucleoprotein complex or large Cas9 expression plasmids. Multi-functional nanovectors enhance genome editing in vivo by overcoming multiple physiological barriers, enabling ligand-targeted cellular uptake, and blood-brain barrier crossing. Chemical engineering can also facilitate viral-based delivery by improving vector internalization, allowing tissue-specific transgene expression, and preventing inactivation of the viral vectors in vivo. This review aims to discuss how chemical engineering has helped improve existing CRISPR applications and enable new technologies for biomedical research. The usefulness, advantages, and molecular action for each chemical engineering approach are also highlighted.

scholarly journals CRISPR-assisted multi-dimensional regulation for fine-tuning gene expression in Bacillus subtilis

2019 ◽  
Vol 47 (7) ◽  
pp. e40-e40 ◽  
Author(s):  
Zhenghui Lu ◽  
Shihui Yang ◽  
Xin Yuan ◽  
Yunyun Shi ◽  
Li Ouyang ◽  
...  
Keyword(s):  
Gene Expression ◽  
Bacillus Subtilis ◽  
Fine Tuning

scholarly journals Fine tuning the glycolytic flux ratio of EP-bifido pathway for mevalonate production by enhancing glucose-6-phosphate dehydrogenase (Zwf) and CRISPRi suppressing 6-phosphofructose kinase (PfkA) in Escherichia coli

2021 ◽  
Vol 20 (1) ◽  
Author(s):  
Ying Li ◽  
He Xian ◽  
Ya Xu ◽  
Yuan Zhu ◽  
Zhijie Sun ◽  
...  
Keyword(s):  
Pentose Phosphate Pathway ◽  
Conversion Rate ◽  
Metabolic Flux ◽  
Flux Ratio ◽  
Reducing Power ◽  
Pentose Phosphate ◽  
Fine Tuning ◽  
High Yield ◽  
Glycolytic Flux ◽  
Acetyl Coa

Abstract Background Natural glycolysis encounters the decarboxylation of glucose partial oxidation product pyruvate into acetyl-CoA, where one-third of the carbon is lost at CO2. We previously constructed a carbon saving pathway, EP-bifido pathway by combining Embden-Meyerhof-Parnas Pathway, Pentose Phosphate Pathway and “bifid shunt”, to generate high yield acetyl-CoA from glucose. However, the carbon conversion rate and reducing power of this pathway was not optimal, the flux ratio of EMP pathway and pentose phosphate pathway (PPP) needs to be precisely and dynamically adjusted to improve the production of mevalonate (MVA). Result Here, we finely tuned the glycolytic flux ratio in two ways. First, we enhanced PPP flux for NADPH supply by replacing the promoter of zwf on the genome with a set of different strength promoters. Compared with the previous EP-bifido strains, the zwf-modified strains showed obvious differences in NADPH, NADH, and ATP synthesis levels. Among them, strain BP10BF accumulated 11.2 g/L of MVA after 72 h of fermentation and the molar conversion rate from glucose reached 62.2%. Second, pfkA was finely down-regulated by the clustered regularly interspaced short palindromic repeats interference (CRISPRi) system. The MVA yield of the regulated strain BiB1F was 8.53 g/L, and the conversion rate from glucose reached 68.7%. Conclusion This is the highest MVA conversion rate reported in shaken flask fermentation. The CRISPRi and promoter fine-tuning provided an effective strategy for metabolic flux redistribution in many metabolic pathways and promotes the chemicals production.

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