Ribosome Binding Site Research Articles

Standardization of Fluorescent Reporter Assays in Synthetic Biology across the Visible Light Spectrum.

In synthetic biology, Fluorescent reporters are frequently used to characterize the expression levels obtained from both genetic parts such as promoters and ribosome binding sites as well as from complex genetic circuits. To this end, plate readers offer an easy and high-throughput way of characterizing both the growth and fluorescence expression levels of cell cultures. However, despite the similar mode of action used in different devices, their output is not comparable due to intrinsic differences in their setup. Additionally, the generated output is expressed using arbitrary units, limiting reliable comparison of results to measurements taken within one single experiment using one specific plate reader, hampering the transferability of data across different plate readers and laboratories. This article presents an easy and accessible calibration method for transforming the device-specific output into a standardized output expressing the amount of fluorescence per well as a known equivalent fluorophore concentration per cell for fluorescent reporters spanning the visible light spectrum. This calibration method follows a 2-fold approach determining both the estimated number of cells and the equivalent chemical fluorophore concentration per well. It will contribute to the comparison of plate reader experiments between different laboratories across the world and will therefore greatly improve the reliability and exchange of both results and genetic parts between research groups.

A smart RBS library and its prediction model for robust and accurate fine-tuning of gene expression in Bacillus species

Bacillus species, such as Bacillus subtilis and Bacillus licheniformis, are important industrial bacteria. However, there is a lack of standardized and predictable genetic tools for convenient and reproducible assembly of genetic modules in Bacillus species to realize their full potential. In this study, we constructed a Ribosome Binding Site (RBS) library in B. licheniformis, which provides incremental regulation of expression levels over a 104-fold range. Additionally, we developed a model to quantify the resulting translation rates. We successfully demonstrated the robust expression of various target genes using the RBS library and showed that the model accurately predicts the translation rates of arbitrary coding genes. Importantly, we also extended the use of the RBS library and prediction model to B. subtilis, B. thuringiensis, and B. amyloliquefacie. The versatility of the RBS library and its prediction model enables quantification of biological behavior, facilitating reliable forward engineering of gene expression.

Construction of cascade circuits for dynamic temporal regulation and its application to PHB production

BackgroundTo maximize the production capacity and yield of microbial cell factories, metabolic pathways are generally modified with dynamic regulatory strategies, which can effectively solve the problems of low biological yield, growth retardation and metabolic imbalance. However, the strategy of dynamic regulating multiple genes in different time and order is still not effectively solved. Based on the quorum-sensing (QS) system and the principle of cascade regulation, we studied the sequence and time interval of gene expression in metabolic pathways.ResultsWe designed and constructed a self-induced dynamic temporal regulatory cascade circuit in Escherichia coli using the QS system and dual regulatory protein cascade and found that the time intervals of the cascade circuits based on the Tra, Las system and the Lux, Tra system reached 200 min and 150 min, respectively. Furthermore, a dynamic temporal regulatory cascade circuit library with time intervals ranging from 110 to 310 min was obtained based on this circuit using promoter engineering and ribosome binding site replacement, which can provide more selective synthetic biology universal components for metabolic applications. Finally, poly-β-hydroxybutyric acid (PHB) production was taken as an example to demonstrate the performance of the cascade circuit library. The content of PHB increased 1.5-fold. Moreover, circuits with different time intervals and different expression orders were found to have different potentials for application in PHB production, and the preferred time-interval circuit strain C2-max was identified by screening.ConclusionsThe self-induced dynamic temporal regulation cascade circuit library can enable the expression of target genes with sequential changes at different times, effectively solving the balance problem between cell growth and product synthesis in two-stage fermentation and expanding the application of dynamic regulatory strategies in the field of metabolic engineering.

De novo design of pimarane diterpenoid compounds as potential alternatives to sarecycline for acne vulgaris treatment

Acne vulgaris is a prevalent skin disorder that affects both adolescents and adults and has a major psychological impact. Antibiotic resistance is one issue that current therapies, including antibiotics, must address. A viable approach is to target Cutibacterium acnes, a crucial bacterium in the development of acne. An antibiotic of the tetracycline class called sarecycline shows efficacy despite resistance and adverse effect issues. The purpose of this study is to develop, assess, and compare the efficacy of Sandracopimar (Pimarane diterpenoids) compounds and Sarecycline in the treatment of acne by targeting the 30S ribosomal subunit of Cutibacterium acnes. Sarecycline’s binding affinity and PheSA score were assessed at the 30S ribosomal subunit binding site of Cutibacterium acnes. There were distinct interactions between Sarecycline and the ribosomal subunit, including hydrophobic and hydrogen bonding. Sarecycline demonstrated a strong binding affinity (-8.2 kcal/mol) and a PheSA score of 0.53184 within the Cutibacterium acnes 30S ribosomal subunit binding site. Sandaracopimar-15-ene-6.beta.,8.beta.-diol exhibited a binding affinity of -7.3 kcal/mol and PheSA score of 0.37252. Compound 1, a novel compound derived from Sandaracopimar-15-ene-6.beta.,8.beta.-diol, showed a slightly higher binding affinity (-8.3 kcal/mol) than Sarecycline. The molecular dynamics simulation results reveal that Compound 1 exhibited stability during a specific phase, indicating favorable binding potential with the Cutibacterium acnes 30S ribosomal subunit drug target. The compound demonstrated structural flexibility, advantageous for molecular interactions. The study indicates that Sandracopimar-derived compounds, including Compound 1, show comparable parameters to Sarecycline, suggesting similar activity in targeting the Cutibacterium acnes 30S ribosomal subunit. These compounds may serve as a potential source of novel anti-acne compounds.

High level food-grade expression of maltogenic amylase in Bacillus subtilis through dal gene auxotrophic selection marker

As a food-safe microorganism, Bacillus subtilis has been widely utilized in the production of food enzyme, where a food-grade expression system without antibiotic is required. However, there is no mature system for such expression, since the recombinant plasmid in existing food-grade expression system is unstable especially in high-density fermentation. In this study, we constructed a food-grade expression system based on the dal gene auxotrophic selection marker. Specifically, maltogenic amylase (AmyM) was expressed in dal deletion strain without antibiotic, yielding an activity of 519 U/mL. To increase the expression of AmyM, the promoter of amyM (gene encoding AmyM) was optimized. Furthermore, we found that excessive expression of dal gene was detrimental to the stability of plasmid, and the ribosome binding site (RBS) of dal was mutated with the reduced synthesis of D-alanine. After that, AmyM activity increased to 1364 U/mL with the 100 % stability of plasmid. The 3-L fermentor cultivation was performed with the highest value ever reported in food-grade microorganisms, an activity of 2388 U/mL, showing the scale-up production capability of this system. Besides, it is also able to apply the system for other food enzymes, which indicating the great generalizability of this system for different application.

Modularized reconstruction of pyrimidine pathway in Escherichia coli for enhanced pyrimidine nucleosides biosynthesis

Pyrimidine is a fundamental component of genetic material and a precursor for synthesis of various antiviral and antitumor drugs. In this study, we constructed a plasmid-free Escherichia coli strain HD11 as a platform for cytidine production. Compared to strain CR23-p21-p31, a higher titer of cytidine was achieved by overexpressing uridylate kinase from Sulfurovum lithotrophicum and the newly discovered cytidine triphosphate synthetase of Aquifex aeolicus in HD11. To enable efficient cytosine biosynthesis, a modularized metabolic pathway was integrated into HD11. Two different pathways based on the type of precursor were compared, and ribosome binding sites were optimized to enhance cytosine production. For pyrimidine ribonucleotide de novo biosynthesis pathway, an enzyme constrained metabolic model was utilized to guide media optimization. Finally, HD11-pDA20 produced 3.6 g/L cytosine and HD11 produced 8.1 g/L cytidine in fed-batch fermentation. The application of an enzyme-constrained metabolic model in cytidine/cytosine producing strain represents a novel approach to modular engineering.

Study on the Genome and Mechanism of Tigecycline Resistance of a Clinical Chryseobacterium indologenes Strain.

Purpose: Chryseobacterium indologenes is a clinically relevant microorganism that has been on the rise, with multidrug-resistant (MDR) strains being reported. C. indologenes carrying tet(X2) has been demonstrated to be resistant to the antibiotic tigecycline, yet, sensitive to all other members of the tetracycline family. This inconsistency in resistance prompts an inquiry into the contribution of tet(X2) to tigecycline resistance in C. indologenes. Materials and Methods: In this study, we report on a comprehensive analysis of the genomic mechanisms underlying tigecycline resistance in a MDR C. indologenes strain (CI3125) that was resistant to tigecycline but sensitive to tetracycline, doxycycline, and minocycline. We used whole-genome sequencing, quantitative reverse transcription PCR, Western blot, antibiotic-degrading tests, and efflux pump inhibiting tests to reveal the mechanism of tigecycline resistance in C. indologenes and elucidate the inconsistency in the antibiotic resistance mechanism for the tetracycline family. Results: Our findings demonstrate that CI3125 carries 60 antibiotic resistance genes distributed on 6 different genetic islands (GIs), with the potential for horizontal transfer. Notably, the tet(X2) gene is located on GI06 of CI3125. Genetic environment analysis of tet(X2) showed that all tet(X2) genes in Flavobacterium and Bacteroides share a conservative and functional ribosome-binding site upstream. Contrary to expectation, our RT-qPCR showed that tet(X2) was not transcribed in CI3125, and Western blot suggested the absence of tet(X2) protein in CI3125. Rather, we demonstrate that minimum inhibitory concentration values for tigecycline decreased two- to eight-folds in the presence of five different efflux pump inhibitors [1-(1-naphthyl- methyl)-piperazine, phenyl-arginine-β-naphthylamide, verapamil, reserpine, and carbonyl cyanide 3-chlorophenylhydrazone]. This finding provides evidence for the involvement of efflux pumps in tigecycline resistance, which is likely to be a universal mechanism among C. indologenes. Our study proposes that the inconsistency in resistance to the tetracycline family in CI3125 may be ascribed to the silence of tet(X2) and the functions of efflux pumps for tigecycline. Conclusions: Overall, our results highlight the importance of genomic approaches in understanding the underlying mechanisms of antibiotic resistance in clinically relevant microorganisms. While tet(X2) in CI3125 is silent, our findings suggest that it may be horizontally spread through GIs. Hence, our findings have significant implications for the management of C. indologenes infections in clinical settings.

Engineering the Maize Root Microbiome: A Rapid MoClo Toolkit and Identification of Potential Bacterial Chassis for Studying Plant-Microbe Interactions.

Sustainably enhancing crop production is a global necessity to meet the escalating demand for staple crops while sustainably managing their associated carbon/nitrogen inputs. Leveraging plant-associated microbiomes is a promising avenue for addressing this demand. However, studying these communities and engineering them for sustainable enhancement of crop production have remained a challenge due to limited genetic tools and methods. In this work, we detail the development of the Maize Root Microbiome ToolKit (MRMTK), a rapid Modular Cloning (MoClo) toolkit that only takes 2.5 h to generate desired constructs (5400 potential plasmids) that replicate and express heterologous genes in Enterobacter ludwigii strain AA4 (Elu), Pseudomonas putida strain AA7 (Ppu), Herbaspirillum robiniae strain AA6 (Hro), Stenotrophomonas maltophilia strain AA1 (Sma), and Brucella pituitosa strain AA2 (Bpi), which comprise a model maize root synthetic community (SynCom). In addition to these genetic tools, we describe a highly efficient transformation protocol (107-109 transformants/μg of DNA) 1 for each of these strains. Utilizing this highly efficient transformation protocol, we identified endogenous Expression Sequences (ES; promoter and ribosomal binding sites) for each strain via genomic promoter trapping. Overall, MRMTK is a scalable and adaptable platform that expands the genetic engineering toolbox while providing a standardized, high-efficiency transformation method across a diverse group of root commensals. These results unlock the ability to elucidate and engineer plant-microbe interactions promoting plant growth for each of the 5 bacterial strains in this study.

An Intelligent Synthetic Bacterium for Chronological Toxicant Detection, Biodegradation, and Its Subsequent Suicide.

Modules, toolboxes, and synthetic biology systems may be designed to address environmental bioremediation. However, weak and decentralized functional modules require complex control. To address this issue, an integrated system for toxicant detection and biodegradation, and subsequent suicide in chronological order without exogenous inducers is constructed. Salicylic acid, a typical pollutant in industrial wastewater, is selected as an example to demonstrate this design. Biosensors are optimized by regulating the expression of receptors and reporters to get 2-fold sensitivity and 6-fold maximum output. Several stationary phase promoters are compared, and promoter Pfic is chosen to express the degradation enzyme. Two concepts for suicide circuits are developed, with the toxin/antitoxin circuit showing potent lethality. The three modules are coupled in a stepwise manner. Detection and biodegradation, and suicide are sequentially completed with partial attenuation compared to pre-integration, except for biodegradation, being improved by the replacements of ribosome binding site. Finally, a long-term stability test reveals that the engineered strain maintained its function for ten generations. The study provides a novel concept for integrating and controlling functional modules that can accelerate the transition of synthetic biology from conceptual to practical applications.

An mTRAN-mRNA interaction mediates mitochondrial translation initiation in plants.

Plant mitochondria represent the largest group of respiring organelles on the planet. Plant mitochondrial messenger RNAs (mRNAs) lack Shine-Dalgarno-like ribosome-binding sites, so it is unknown how plant mitoribosomes recognize mRNA. We show that "mitochondrial translation factors" mTRAN1 and mTRAN2 are land plant-specific proteins, required for normal mitochondrial respiration chain biogenesis. Our studies suggest that mTRANs are noncanonical pentatricopeptide repeat (PPR)-like RNA binding proteins of the mitoribosomal "small" subunit. We identified conserved Adenosine (A)/Uridine (U)-rich motifs in the 5' regions of plant mitochondrial mRNAs. mTRAN1 binds this motif, suggesting that it is a mitoribosome homing factor to identify mRNAs. We demonstrate that mTRANs are likely required for translation of all plant mitochondrial mRNAs. Plant mitochondrial translation initiation thus appears to use a protein-mRNA interaction that is divergent from bacteria or mammalian mitochondria.

Structure and function analysis of a type III preQ1-I riboswitch from Escherichia coli reveals direct metabolite sensing by the Shine-Dalgarno sequence

Riboswitches are small noncoding RNAs found primarily in the 5' leader regions of bacterial messenger RNAs where they regulate expression of downstream genes in response to binding one or more cellular metabolites. Such noncoding RNAs are often regulated at the translation level, which is thought to be mediated by the accessibility of the Shine-Dalgarno sequence (SDS) ribosome-binding site. Three classes (I-III) of prequeuosine1 (preQ1)-sensing riboswitches are known that control translation. Class I is divided into three subtypes (types I-III) that have diverse mechanisms of sensing preQ1, which is involved in queuosine biosynthesis. To provide insight into translation control, we determined a 2.30 Å-resolution cocrystal structure of a class I type III preQ1-sensing riboswitch identified in Escherichia coli (Eco) by bioinformatic searches. The Eco riboswitch structure differs from previous preQ1 riboswitch structures because it has the smallest naturally occurring aptamer and the SDS directly contacts the preQ1 metabolite. We validated structural observations using surface plasmon resonance and invivo gene-expression assays, which showed strong switching in live E.coli. Our results demonstrate that the Eco riboswitch is relatively sensitive to mutations that disrupt noncanonical interactions that form the pseudoknot. In contrast to type II preQ1 riboswitches, a kinetic analysis showed that the type III Eco riboswitch strongly prefers preQ1 over the chemically similar metabolic precursor preQ0. Our results reveal the importance of noncanonical interactions in riboswitch-driven gene regulation and the versatility of the class I preQ1 riboswitch pseudoknot as a metabolite-sensing platform that supports SDS sequestration.

Rational metabolic engineering of Corynebacterium glutamicum for efficient synthesis of L-glutamate

L-glutamic acid is the world's largest bulk amino acid product that is widely used in the food, pharmaceutical and chemical industries. Using Corynebacterium glutamicum G01 as the starting strain, the fermentation by-product alanine content was firstly reduced by knocking out the gene encoding alanine aminotransferase (alaT), a major by-product related to alanine synthesis. Secondly, since the α-ketoglutarate node carbon flow plays an important role in glutamate synthesis, the ribosome-binding site (RBS) sequence optimization was used to reduce the activity of α-ketoglutarate dehydrogenase and enhance the glutamate anabolic flow. The endogenous conversion of α-ketoglutarate to glutamate was also enhanced by screening different glutamate dehydrogenase. Subsequently, the glutamate transporter was rationally desgined to improve the glutamate efflux capacity. Finally, the fermentation conditions of the strain constructed using the above strategy were optimized in 5 L fermenters by a gradient temperature increase combined with a batch replenishment strategy. The glutamic acid production reached (135.33±4.68) g/L, which was 41.2% higher than that of the original strain (96.53±2.32) g/L. The yield was 55.8%, which was 11.6% higher than that of the original strain (44.2%). The combined strategy improved the titer and the yield of glutamic acid, which provides a reference for the metabolic modification of glutamic acid producing strains.

The small non-coding RNA B11 regulates multiple facets of Mycobacterium abscessus virulence.

Mycobacterium abscessus causes severe disease in patients with cystic fibrosis. Little is known in M. abscessus about the roles of small regulatory RNAs (sRNA) in gene regulation. We show that the sRNA B11 controls gene expression and virulence-associated phenotypes in this pathogen. B11 deletion from the smooth strain ATCC_19977 produced a rough strain, increased pro-inflammatory signaling and virulence in multiple infection models, and increased resistance to antibiotics. Examination of clinical isolate cohorts identified isolates with B11 mutations or reduced expression. We used RNAseq and proteomics to investigate the effects of B11 on gene expression and test the impact of mutations found in clinical isolates. Over 200 genes were differentially expressed in the deletion mutant. Strains with the clinical B11 mutations showed expression trends similar to the deletion mutant, suggesting partial loss of function. Among genes upregulated in the B11 mutant, there was a strong enrichment for genes with B11-complementary sequences in their predicted ribosome binding sites (RBS), consistent with B11 functioning as a negative regulator that represses translation via base-pairing to RBSs. Comparing the proteomes similarly revealed that upregulated proteins were strongly enriched for B11-complementary sequences. Intriguingly, genes upregulated in the absence of B11 included components of the ESX-4 secretion system, critical for M. abscessus virulence. Many of these genes had B11-complementary sequences at their RBSs, which we show is sufficient to mediate repression by B11 through direct binding. Altogether, our data show that B11 acts as a direct negative regulator and mediates (likely indirect) positive regulation with pleiotropic effects on gene expression and clinically important phenotypes in M. abscessus. The presence of hypomorphic B11 mutations in clinical strains is consistent with the idea that lower B11 activity may be advantageous for M. abscessus in some clinical contexts. This is the first report on an sRNA role in M. abscessus.

New Resistance Mutations Linked to Decreased Susceptibility to Solithromycin in Streptococcus pneumoniae Revealed by Chemogenomic Screens.

Two strains of Streptococcus pneumoniae, one expressing the methyltransferase Erm(B) and the other negative for erm(B), were selected for solithromycin resistance in vitro either with direct drug selection or with chemical mutagenesis followed by drug selection. We obtained a series of mutants that we characterized by next-generation sequencing. We found mutations in various ribosomal proteins (L3, L4, L22, L32, and S4) and in the 23S rRNA. We also found mutations in subunits of the phosphate transporter, in the DEAD box helicase CshB, and in the erm(B)L leader peptide. All mutations were shown to decrease solithromycin susceptibility when transformed into sensitive isolates. Some of the genes derived from our in vitro screens were found to be mutated also in clinical isolates with decreased susceptibility to solithromycin. While many mutations were in coding sequences, some were found in regulatory regions. These included novel phenotypic mutations in the intergenic regions of the macrolide resistance locus mef(E)/mel and in the vicinity of the ribosome binding site of erm(B). Our screens highlighted that macrolide-resistant S. pneumoniae can easily acquire resistance to solithromycin, and they revealed many new phenotypic mutations.

Highly Efficient Synthesis of Rare Sugars from Glycerol in Endotoxin-Free ClearColi by Fermentation.

Rare sugars possess potential applications as low-calorie sweeteners, especially for anti-obesity and anti-diabetes. In this study, a fermentation biosystem based on the "DHAP-dependent aldolases strategy" was established for D-allulose and D-sorbose production from glycerol in endotoxin-free ClearColi BL21 (DE3). Several engineering strategies were adopted to enhance rare sugar production. Firstly, the combination of different plasmids for aldO, rhaD, and yqaB expression was optimized. Then, the artificially constructed ribosomal binding site (RBS) libraries of aldO, rhaD, and yqaB genes were assembled individually and combinatorially. In addition, a peroxidase was overexpressed to eliminate the damage or toxicity from hydrogen peroxide generated by alditol oxidase (AldO). Finally, stepwise improvements in rare sugar synthesis were elevated to 15.01 g/L with a high yield of 0.75 g/g glycerol in a 3 L fermenter. This research enables the effective production of rare sugars from raw glycerol in high yields.

Linking folding dynamics and function of SAM/SAH riboswitches at the single molecule level.

Riboswitches are regulatory elements found in bacterial mRNAs that control downstream gene expression through ligand-induced conformational changes. Here, we used single-molecule FRET to map the conformational landscape of the translational SAM/SAH riboswitch and probe how co-transcriptional ligand-induced conformational changes affect its translation regulation function. Riboswitch folding is highly heterogeneous, suggesting a rugged conformational landscape that allows for sampling of the ligand-bound conformation even in the absence of ligand. The addition of ligand shifts the landscape, favoring the ligand-bound conformation. Mutation studies identified a key structural element, the pseudoknot helix, that is crucial for determining ligand-free conformations and their ligand responsiveness. We also investigated ribosomal binding site accessibility under two scenarios: pre-folding and co-transcriptional folding. The regulatory function of the SAM/SAH riboswitch involves kinetically favoring ligand binding, but co-transcriptional folding reduces this preference with a less compact initial conformation that exposes the Shine-Dalgarno sequence and takes min to redistribute to more compact conformations of the pre-folded riboswitch. Such slow equilibration decreases the effective ligand affinity. Overall, our study provides a deeper understanding of the complex folding process and how the riboswitch adapts its folding pattern in response to ligand, modulates ribosome accessibilityand the role of co-transcriptional folding in these processes.

Rapid construction of E.coli chassis with genome multi-position integration of isopentenol utilization pathway for efficient and stable terpenoid accumulation.

The isopentenol utilization pathway (IUP) is potential in terpenoids synthesis. This study aimed to construct IUP-employed E. coli chassis for stably synthesizing terpenoids. As to effectiveness, promotor engineering strategy was employed to regulate IUP expression level, while ribosome-binding site (RBS) library of the key enzyme was constructed for screening the optimal RBS, followed by optimization of concentration of inducer and substrates, the titer of reporting production, lycopene, from 0.087 to 8.67 mg/OD600 . As about stability, the IUP expression cassette was integrated into the genome through transposition tool based on CRISPR-associated transposases. Results showed that the strain with 13 copies produced 1.78-fold lycopene titer that of the controlled strain with IUP-harbored plasmid, and it exhibited stable expression after ten successions while the plasmid loss was observed in the controlled strain in the 3rd succession. This strategy provides valuable information for rapid construction of highly effective and stable chassis employing IUP for terpenoids production. This article is protected by copyright. All rights reserved.

A robust genetic toolbox for fine-tuning gene expression in the CO2-Fixing methanogenic archaeon Methanococcus maripaludis

Libraries of well-characterized genetic elements for fine-tuning gene expression are essential for biological and biotechnological research and applications. The fast-growing and genetically tractable methanogen, Methanococcus maripaludis, is a promising host organism for biotechnological conversion of carbon dioxide and renewable hydrogen into fuels and value-added products, as well as fundamental biological studies of archaea. However, the lack of molecular tools for gene expression has hindered its application as a workhorse to fine-tune gene and metabolic pathway expressions. In this study, we developed a genetic toolbox, including libraries of promoters, ribosome binding sites (RBS), and neutral sites for chromosomal integration, to facilitate precise gene expression in M. maripaludis. We generated a promoter library consisting of 81 constitutive promoters with expression strengths spanning a ∼104-fold dynamic range. Importantly, we identified a base composition rule for strong archaeal promoters and successfully remodeled weak promoters, enhancing their activities by up to 120-fold. We also established an RBS library containing 42 diverse RBS sequences with translation strengths covering a ∼100-fold dynamic range. Additionally, we identified eight neutral sites and developed a one-step, Cas9-based marker-less knock-in approach for chromosomal integration. We successfully applied the characterized promoter and RBS elements to significantly improve recombinant protein expression by 41-fold and modulate essential gene expression to generate corresponding physiological changes in M. maripaludis. Therefore, this work establishes a solid foundation for utilizing this autotrophic methanogen as an ideal workhorse for archaeal biology and biotechnological studies and applications.

Metabolic engineering of Escherichia coli for efficient production of linalool from biodiesel-derived glycerol by targeting cofactors regeneration and reducing acetate accumulation

Linalool is a valuable monoterpenoid compound commercially used in cosmetics and personal care industry as well as a ‘drop-in’ biofuel. Direct plant extraction or chemical synthesis of linalool suffers from insufficient abundance in plant and inefficient purification procedures. Herein, a powerful green cell factory has been developed to meet the increasing demands for more sustainable and efficient production of this terpene alcohol. The de novo production of linalool from biodiesel-derived glycerol was achieved by engineered Escherichia coli harboring the exogenous mevalonate pathway followed by coupling with geranyl diphosphate synthase and linalool synthase (LIS). Then, the Mentha aquatica LIS showing the most efficient linalool biosynthesis was identified, and subsequently tuned by ribosomal binding site (RBS) engineering and site-directed mutagenesis to facilitate linalool production. A further engineered strain equipped with an engineered NADP+-dependent pyruvate dehydrogenase complex (PDH) and phosphoketolase bypass, remarkably decreasing the by-products formation together with boosting substrate utilization efficiency. A final titer of 1.07 g/L linalool with a yield of 0.10 g/g glycerol was gained in flask culture and further increase to 4.16 g/L in 2-L fed-batch fermentation, which is the highest reported linalool titer and yield in E. coli to the best of our knowledge. In addition, the engineering strategies established here not only lay a potential production platform for linalool and its derivatives, but also provide references to minimize by-products secretion and improve carbon use efficiency.

Enhanced extracellular production of raw starch-degrading α-amylase in Bacillus subtilis through expression regulatory element modification and fermentation optimization

BackgroundRaw starch-degrading α-amylase (RSDA) can hydrolyze raw starch at moderate temperatures, thus contributing to savings in starch processing costs. However, the low production level of RSDA limits its industrial application. Therefore, improving the extracellular expression of RSDA in Bacillus subtilis, a commonly used industrial expression host, has great value.ResultsIn this study, the extracellular production level of Pontibacillus sp. ZY raw starch-degrading α-amylase (AmyZ1) in B. subtilis was enhanced by expression regulatory element modification and fermentation optimization. As an important regulatory element of gene expression, the promoter, signal peptide, and ribosome binding site (RBS) sequences upstream of the amyZ1 gene were sequentially optimized. Initially, based on five single promoters, the dual-promoter Pveg-PylB was constructed by tandem promoter engineering. Afterward, the optimal signal peptide SPNucB was obtained by screening 173 B. subtilis signal peptides. Then, the RBS sequence was optimized using the RBS Calculator to obtain the optimal RBS1. The resulting recombinant strain WBZ-VY-B-R1 showed an extracellular AmyZ1 activity of 4824.2 and 41251.3 U/mL during shake-flask cultivation and 3-L fermenter fermentation, which were 2.6- and 2.5-fold greater than those of the original strain WBZ-Y, respectively. Finally, the extracellular AmyZ1 activity of WBZ-VY-B-R1 was increased to 5733.5 U/mL in shake flask by optimizing the type and concentration of carbon source, nitrogen source, and metal ions in the fermentation medium. On this basis, its extracellular AmyZ1 activity was increased to 49082.1 U/mL in 3-L fermenter by optimizing the basic medium components as well as the ratio of carbon and nitrogen sources in the feed solution. This is the highest production level reported to date for recombinant RSDA production.ConclusionsThis study represents a report on the extracellular production of AmyZ1 using B. subtilis as a host strain, and achieved the current highest expression level. The results of this study will lay a foundation for the industrial application of RSDA. In addition, the strategies employed here also provide a promising way for improving other protein production in B. subtilis.