Multiplexed in-situ mutagenesis driven by a dCas12a-based dual-function base editor.

Mutagenesis driving genetic diversity is vital for understanding and engineering biological systems. However, the lack of effective methods to generate in-situ mutagenesis in multiple genomic loci combinatorially limits the study of complex biological functions. Here, we design and construct MultiduBE, a dCas12a-based multiplexed dual-function base editor, in an all-in-one plasmid for performing combinatorial in-situ mutagenesis. Two synthetic effectors, duBE-1a and duBE-2b, are created by amalgamating the functionalities of cytosine deaminase (from hAPOBEC3A or hAID*Δ ), adenine deaminase (from TadA9), and crRNA array processing (from dCas12a). Furthermore, introducing the synthetic separator Sp4 minimizes interference in the crRNA array, thereby facilitating multiplexed in-situ mutagenesis in both Escherichia coli and Bacillus subtilis. Guided by the corresponding crRNA arrays, MultiduBE is successfully employed for cell physiology reprogramming and metabolic regulation. A novel mutation conferring streptomycin resistance has been identified in B. subtilis and incorporated into the mutant strains with multiple antibiotic resistance. Moreover, surfactin and riboflavin titers of the combinatorially mutant strains improved by 42% and 15-fold, respectively, compared with the control strains with single gene mutation. Overall, MultiduBE provides a convenient and efficient way to perform multiplexed in-situ mutagenesis.

Engineered bacterial orthogonal DNA replication system for continuous evolution.

Continuous evolution can generate biomolecules for synthetic biology and enable evolutionary investigation. The orthogonal DNA replication system (OrthoRep) in yeast can efficiently mutate long DNA fragments in an easy-to-operate manner. However, such a system is lacking in bacteria. Therefore, we developed a bacterial orthogonal DNA replication system (BacORep) for continuous evolution. We achieved this by harnessing the temperate phage GIL16 DNA replication machinery in Bacillus thuringiensis with an engineered error-prone orthogonal DNA polymerase. BacORep introduces all 12 types of nucleotide substitution in 15-kilobase genes on orthogonally replicating linear plasmids with a 6,700-fold higher mutation rate than that of the host genome, the mutation rate of which is unchanged. Here we demonstrate the utility of BacORep-based continuous evolution by generating strong promoters applicable to model bacteria, Bacillus subtilis and Escherichia coli, and achieving a 7.4-fold methanol assimilation increase in B. thuringiensis. BacORep is a powerful tool for continuous evolution in prokaryotic cells.

CRISPR-dCas12a-mediated genetic circuit cascades for multiplexed pathway optimization.

The production efficiency of microbial cell factories is sometimes limited by the lack of effective methods to regulate multiple targets in a coordinated manner. Here taking the biosynthesis of glucosamine-6-phosphate (GlcN6P) in Bacillus subtilis as an example, a 'design-build-test-learn' framework was proposed to achieve efficient multiplexed optimization of metabolic pathways. A platform strain was built to carry biosensor signal-amplifying circuits and two genetic regulation circuits. Then, a synthetic CRISPR RNA array blend for boosting and leading (ScrABBLE) device was integrated into the platform strain, which generated 5,184 combinatorial assemblies targeting three genes. The best GlcN6P producer was screened and engineered for the synthesis of valuable pharmaceuticals N-acetylglucosamine and N-acetylmannosamine. The N-acetylglucosamine titer reached 183.9 g liter-1 in a 15-liter bioreactor. In addition, the potential generic application of the ScrABBLE device was also verified using three fluorescent proteins as a case study.

A pathway independent multi-modular ordered control system based on thermosensors and CRISPRi improves bioproduction in Bacillus subtilis.

Dynamic regulation is an effective strategy for control of gene expression in microbial cell factories. In some pathway contexts, several metabolic modules must be controlled in a time dependent or ordered manner to maximize production, while the creation of genetic circuits with ordered regulation capacity still remains a great challenge. In this work, we develop a pathway independent and programmable system that enables multi-modular ordered control of metabolism in Bacillus subtilis. First, a series of thermosensors were created and engineered to expand their thresholds. Then we designed single-input-multi-output circuits for ordered control based on the use of thermosensors with different transition points. Meanwhile, a repression circuit was constructed by combining CRISPRi-based NOT gates. As a proof-of-concept, these genetic circuits were applied for multi-modular ordered control of 2'-fucosyllactose (2'-FL) biosynthesis, resulting in a production of 1839.7 mg/l in shake flask, which is 5.16-times that of the parental strain. In a 5-l bioreactor, the 2'-FL titer reached 28.2 g/l with down-regulation of autolysis. Taken together, this work provides programmable and versatile thermosensitive genetic toolkits for dynamic regulation in B. subtilis and a multi-modular ordered control framework that can be used to improve metabolic modules in other chassis cells and for other compounds.

etiBsu1209: A comprehensive multiscale metabolic model for Bacillus subtilis.

Genome-scale metabolic models (GEMs) have been widely used to guide the computational design of microbial cell factories, and to date, seven GEMs have been reported for Bacillus subtilis, a model gram-positive microorganism widely used in bioproduction of functional nutraceuticals and food ingredients. However, none of them are widely used because they often lead to erroneous predictions due to their low predictive power and lack of information on regulatory mechanisms. In this work, we constructed a new version of GEM for B. subtilis (iBsu1209), which contains 1209 genes, 1595 metabolites, and 1948 reactions. We applied machine learning to fill gaps, which formed a relatively complete metabolic network able to predict with high accuracy (89.3%) the growth of 1209 mutants under 12 different culture conditions. In addition, we developed a visualization and code-free software, Model Tool, for multiconstraints model reconstruction and analysis. We used this software to construct etiBsu1209, a multiscale model that integrates enzymatic constraints, thermodynamic constraints, and transcriptional regulatory networks. Furthermore, we used etiBsu1209 to guide a metabolic engineering strategy (knocking out fabI and yfkN genes) for the overproduction of nutraceutical menaquinone-7, and the titer increased to 153.94 mg/L, 2.2-times that of the parental strain. To the best of our knowledge, etiBsu1209 is the first comprehensive multiscale model for B. subtilis and can serve as a solid basis for rational computational design of B. subtilis cell factories for bioproduction.

Analysis of acid-tolerance mechanism based on membrane microdomains in Saccharomyces cerevisiae.

BACKGROUND: Saccharomyces cerevisiae has been used in the biosynthesis of acid products such as organic acids owing to its acid tolerance. Improving the acid tolerance of S. cerevisiae is beneficial for expanding its application range. Our previous study isolated the TAMC strain that was tolerant to a pH 2.3 through adaptive laboratory evolution; however, its mechanism underlying tolerance to low pH environment remains unclear. RESULTS: In this study, through visual observation and order analysis of plasma membrane and membrane microdomains, we revealed that the membrane microdomains of TAMC strain play an indispensable role in acid tolerance. Transcriptomic analysis showed an increase in the expression of genes related to key components of membrane microdomains in TAMC strain. Furthermore, an obvious reduction was observed in the acid tolerance of the strain with sterol C-24 methyltransferase encoding gene ERG6 knockout for inhibiting membrane microdomain formation. Finally, colocalization analysis of H+-ATPase PMA1 and plasma membrane protein PMP1 showed that disruption of membrane microdomains could inhibit the formation of the H+-ATPase complex. CONCLUSIONS: Membrane microdomains could provide a platform for forming H+-ATPase complexes to facilitate intracellular H+ homeostasis, and thereby improve cell acid resistance. This study proposed a novel acid tolerance mechanism, providing a new direction for the rational engineering of acid-tolerant strains.

Combinatorial metabolic engineering enables high yield production of α-arbutin from sucrose by biocatalysis.

α-Arbutin has been widely used as a skin-whitening ingredient. Previously, we successfully produced α-arbutin via whole-cell biocatalysis and found that the conversion rate of sucrose to α-arbutin was low (~45%). To overcome this issue, herein, we knocked out the genes of enzymes related to the sucrose hydrolysis, including sacB, sacC, levB, and sacA. The sucrose consumption was reduced by 17.4% in 24 h, and the sucrose conversion rate was increased to 51.5%. Furthermore, we developed an inducible protein degradation system with Lon protease isolated from Mesoplasma florum (MfLon) and proteolytic tag to control the PfkA activity, so that more fructose-6-phosphate (F6P) can be converted into glucose-1-phosphate (Glc1P) for α-arbutin synthesis, which can reduce the addition of sucrose and increase the sucrose conversion efficiency. Finally, the pathway of F6P to Glc1P was enhanced by integrating another copy of glucose 6-phosphate isomerase (Pgi) and phosphoglucomutase (PgcA); a high α-arbutin titer (~120 g/L) was obtained. The sucrose conversion rate was increased to 60.4% (mol/mol). In this study, the substrate utilization rate was boosted due to the attenuation of its hydrolysis and the assistance of the intracellular enzymes that converted the side product back into the substrate for α-arbutin synthesis. This strategy provides a new idea for the whole-cell biocatalytic synthesis of other products using sucrose as substrate, especially valuable glycosides.Key points The genes of sucrose metabolic pathway were knocked out to reduce the sucrose consumption. The by-product fructose was reused to synthesize α-arbutin. The optimized whole-cell system improved sucrose conversion by 15.3%.

Combinatorial pathway engineering of Bacillus subtilis for production of structurally defined and homogeneous chitooligosaccharides.

Chitooligosaccharides (COSs) have a widespread range of biological functions and an incredible potential for various pharmaceutical and agricultural applications. Although several physical, chemical, and biological techniques have been reported for COSs production, it is still a challenge to obtain structurally defined COSs with defined polymerization (DP) and acetylation patterns, which hampers the specific characterization and application of COSs. Herein, we achieved the de novo production of structurally defined COSs using combinatorial pathway engineering in Bacillus subtilis. Specifically, the COSs synthase NodC from Azorhizobium caulinodans was overexpressed in B. subtilis, leading to 30 ± 0.86 mg/L of chitin oligosaccharides (CTOSs), the homo-oligomers of N-acetylglucosamine (GlcNAc) with a well-defined DP lower than 6. Then introduction of a GlcNAc synthesis module to promote the supply of the sugar acceptor GlcNAc, reduced CTOSs production, which suggested that the activity of COSs synthase NodC and the supply of sugar donor UDP-GlcNAc may be the limiting steps for CTOSs synthesis. Therefore, 6 exogenous COSs synthase candidates were examined, and the nodCM from Mesorhizobium loti yielded the highest CTOSs titer of 560 ± 16 mg/L. Finally, both the de novo pathway and the salvage pathway of UDP-GlcNAc were engineered to further promote the biosynthesis of CTOSs. The titer of CTOSs in 3-L fed-batch bioreactor reached 4.82 ± 0.11 g/L (85.6% CTOS5, 7.5% CTOS4, 5.3% CTOS3 and 1.6% CTOS2), which was the highest ever reported. This is the first report proving the feasibility of the de novo production of structurally defined CTOSs by synthetic biology, and provides a good starting point for further engineering to achieve the commercial production.

Secretion of Phospholipase Dδ Functions as a Regulatory Mechanism in Plant Innate Immunity.

Plant phospholipase Ds (PLDs), essential regulators of phospholipid signaling, function in multiple signal transduction cascades; however, the mechanisms regulating PLDs in response to pathogens remain unclear. Here, we found that Arabidopsis (Arabidopsis thaliana) PLDδ accumulated in cells at the entry sites of the barley powdery mildew fungus, Blumeria graminis f. sp hordei Using fluorescence recovery after photobleaching and single-molecule analysis, we observed higher PLDδ density in the plasma membrane after chitin treatment; PLDδ also underwent rapid exocytosis. Fluorescence resonance energy transfer with fluorescence lifetime imaging microscopy showed that the interaction between PLDδ and the microdomain marker AtREMORIN1.3 (AtREM1.3) increased in response to chitin, indicating that exocytosis facilitates rapid, efficient sorting of PLDδ into microdomains upon pathogen stimulus. We further unveiled a trade-off between brefeldin A (BFA)-resistant and -sensitive pathways in secretion of PLDδ under diverse conditions. Upon pathogen attack, PLDδ secretion involved syntaxin-associated VAMP721/722-mediated exocytosis sensitive to BFA. Analysis of phosphatidic acid (PA), hydrogen peroxide, and jasmonic acid (JA) levels and expression of related genes indicated that the relocalization of PLDδ is crucial for its activation to produce PA and initiate reactive oxygen species and JA signaling pathways. Together, our findings revealed that the translocation of PLDδ to papillae is modulated by exocytosis, thus triggering PA-mediated signaling in plant innate immunity.plantcell;31/12/3015/FX1F1fx1.

Gallium nitride-based resonant cavity light-emitting diode with single-longitudinal-mode emission.

A novel, to the best of our knowledge, gallium nitride (GaN)-based resonant cavity light-emitting diode (RCLED) with single-longitudinal-mode light emission was demonstrated. A Ta2O5/SiO2 dielectric distributed Bragg reflector (DBR) with a filter-film structure was adopted as the top mirror. In contrast to the flat-topped reflectivity spectrum of the conventional high-reflective-structure DBR, for this filter-film-structure DBR, there is a light-transmitting concave band on the reflectivity spectrum. Owing to the modulation effect of this band on the output light, a single-longitudinal-mode light emission with a full width at half maximum as low as 0.63 nm was realized. Furthermore, the novel RCLED exhibited better wavelength stability. With an increase in the injection current from 50 to 500 mA, the redshift of the emission peak was only 0.2 nm. This novel RCLED with ultra-narrowband emission has a high potential for application in optical communication systems and optical fiber sensing applications.

Engineered yeast for efficient de novo synthesis of 7-dehydrocholesterol.

The synthesis of vitamin D3 precursor 7-dehydrocholesterol (7-DHC) by microbial fermentation has much attracted attention owing to its advantages of environmental protection. In this study, Saccharomyces cerevisiae was engineered for a de novo biosynthesis of 7-DHC. First, seven essential genes (six endogenous genes and one heterologous gene) were overexpressed, and the ROX1 gene (heme-dependent repressor of hypoxic genes) was knocked out. The resulting strain produced 82.6 mg/L 7-DHC from glucose. Then, we predicted five gene knockout targets for 7-DHC overproduction by the reconstruction of genome-scale metabolic model. GDH1 gene knockout increased the 7-DHC titer from 82.6 to 101.5 mg/L, and the specific growth rate of the ΔGDH1 mutant was also increased by 28%. Next, Ty1 transposon in S. cerevisiae was applied to increase the copies of the ERG1 gene and DHCR24 gene, resulting in a 120% increase in 7-DHC titer to 223.3 mg/L. Besides, to optimize the metabolic flux distribution, Clustered Regularly Interspaced Short Palindromic Repeats interference (CRISPRi) system was used to dynamically inhibit the competitive pathway, and the best binding site of ERG6 (delta (24)-sterol C-methyltransferase) promoter was screened out. The OD600 value of ERG6 regulated cells increased by 43% than knocking out ERG6 directly, and 7-DHC titer increased to 365.5 mg/L in a shake flask. Finally, the 7-DHC titer reached 1328 mg/L in 3-L bioreactor and the specific titer of 7-DHC reached up to 114.7 mg/g dry cell weight). Overall, this study constructed a yeast chassis for the highly efficient production of 7-DHC by systems metabolic engineering.

Compartmentalization and transporter engineering strategies for terpenoid synthesis.

Microbial cell factories for terpenoid synthesis form a less expensive and more environment-friendly approach than chemical synthesis and extraction, and are thus being regarded as mainstream research recently. Organelle compartmentalization for terpenoid synthesis has received much attention from researchers owing to the diverse physiochemical characteristics of organelles. In this review, we first systematically summarized various compartmentalization strategies utilized in terpenoid production, mainly plant terpenoids, which can provide catalytic reactions with sufficient intermediates and a suitable environment, while bypassing competing metabolic pathways. In addition, because of the limited storage capacity of cells, strategies used for the expansion of specific organelle membranes were discussed. Next, transporter engineering strategies to overcome the cytotoxic effects of terpenoid accumulation were analyzed. Finally, we discussed the future perspectives of compartmentalization and transporter engineering strategies, with the hope of providing theoretical guidance for designing and constructing cell factories for the purpose of terpenoid production.

Engineered Bacillus subtilis for the de novo production of 2'-fucosyllactose.

BACKGROUND: The most abundant human milk oligosaccharide in breast milk, 2'-fucosyllactose (2'-FL), has been approved as an additive to infant formula due to its multifarious nutraceutical and pharmaceutical functions in promoting neonate health. However, the low efficiency of de novo synthesis limits the cost-efficient bioproduction of 2'-FL. RESULTS: This study achieved 2'-FL de novo synthesis in a generally recognized as safe (GRAS) strain Bacillus subtilis. First, a de novo biosynthetic pathway for 2'-FL was introduced by expressing the manB, manC, gmd, wcaG, and futC genes from Escherichia coli and Helicobacter pylori in B. subtilis, resulting in 2'-FL production of 1.12 g/L. Subsequently, a 2'-FL titer of 2.57 g/L was obtained by reducing the competitive lactose consumption, increasing the regeneration of the cofactor guanosine-5'-triphosphate (GTP), and enhancing the supply of the precursor mannose-6-phosphate (M6P). By replacing the native promoter of endogenous manA gene (encoding M6P isomerase) with a constitutive promoter P7, the 2'-FL titer in shake flask reached 18.27 g/L. The finally engineered strain BS21 could produce 88.3 g/L 2'-FL with a yield of 0.61 g/g lactose in a 3-L bioreactor, without the addition of antibiotics and chemical inducers. CONCLUSIONS: The efficient de novo synthesis of 2'-FL can be achieved by the engineered B. subtilis, paving the way for the large-scale bioproduction of 2'-FL titer in the future.

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

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.

Synergistic improvement of N-acetylglucosamine production by engineering transcription factors and balancing redox cofactors.

The regulation of single gene transcription level in the metabolic pathway is often failed to significantly improve the titer of the target product, and even leads to the imbalance of carbon/nitrogen metabolic network and cofactor network. Global transcription machinery engineering (gTME) can activate or inhibit the synergistic expression of multiple genes in specific metabolic pathways, so transcription factors with specific functions can be expressed according to different metabolic regulation requirements, thus effectively increasing the synthesis of target metabolites. In addition, maintaining intracellular redox balance through cofactor engineering can realize the self-balance of cofactors and promote the efficient synthesis of target products. In this study, we rebalanced the central carbon/nitrogen metabolism and redox metabolism of Corynebacterium glutamicum S9114 by gTME and redox cofactors engineering to promote the production of the nutraceutical N-acetylglucosamine (GlcNAc). Firstly, it was found that the overexpression of the transcription factor RamA can promote GlcNAc synthesis, and the titer was further improved to 16 g/L in shake flask by using a mutant RamA (RamAM). Secondly, a CRISPR interference (CRISPRi) system based on dCpf1 was developed and used to inhibit the expression of global negative transcriptional regulators of GlcNAc synthesis, which promoted the GlcNAc titer to 27.5 g/L. Thirdly, the cofactor specificity of the key enzymes in GlcNAc synthesis pathway was changed by rational protein engineering, and the titer of GlcNAc in shake flask was increased to 36.9 g/L. Finally, the production of GlcNAc was scaled up in a 50-L fermentor, and the titer reached 117.1 ± 1.9 g/L, which was 6.62 times that of the control group (17.7 ± 0.4 g/L), and the yield was increased from 0.19 g/g to 0.31 g/g glucose. The results obtained here highlight the importance of engineering the global regulation of central carbon/nitrogen metabolism and redox metabolism to improve the production performance of microbial cell factories.

The elucidation of phosphosugar stress response in Bacillus subtilis guides strain engineering for high N-acetylglucosamine production.

Bacillus subtilis is a preferred microbial host for the industrial production of nutraceuticals and a promising candidate for the synthesis of functional sugars, such as N-acetylglucosamine (GlcNAc). Previously, a GlcNAc-overproducer B. subtilis SFMI was constructed using glmS ribozyme dual-regulatory tool. Herein, we further engineered to enhance carbon flux from glucose towards GlcNAc synthesis. As a result, the increased flux towards GlcNAc synthesis triggered phosphosugar stress response, which caused abnormal cell growth. Unfortunately, the mechanism of phosphosugar stress response had not been elucidated in B. subtilis. To reveal the stress mechanism and overcome its negative effect in bioproduction, we performed comparative transcriptome analysis. The results indicate that cells slow glucose utilization by repression of glucose import and accelerate catabolic reactions of phosphosugar. To verify these results, we overexpressed the phosphatase YwpJ, which relieved phosphosugar stress and allowed us to identify the enzyme responsible for GlcNAc synthesis from GlcNAc 6-phosphate. In addition, the deletion of nagBB and murQ, responsible for GlcNAc precursor degradation, further improved GlcNAc synthesis. The best engineered strain, B. subtilis FMIP34, increased GlcNAc titer from 11.5 to 26.1 g/L in shake flasks and produced 87.5 g/L GlcNAc in 30-L fed-batch bioreactor. Our results not only elucidate, for the first time, the phosphosugar stress response mechanism in B. subtilis, but also demonstrate how the combination of rational metabolic engineering with novel insights into physiology and metabolism allows the construction of highly efficient microbial cell factories for the production of high-value chemicals.

Semi-rational design of L-amino acid deaminase for production of pyruvate and D-alanine by Escherichia coli whole-cell biocatalyst.

In our previous study, one-step pyruvate and D-alanine production from D,L-alanine by a whole-cell biocatalyst Escherichia coli expressing L-amino acid deaminase (Pm1) derived from Proteus mirabilis was investigated. However, due to the low catalytic efficiency of Pm1, the pyruvate titer was relatively low. Here, semi-rational design based on site-directed saturation mutagenesis was carried out to improve the catalytic efficiency of Pm1. A novel high-throughput screening (HTS) method for pyruvate based on 2,4-dinitrophenylhydrazine indicator was then established. The catalytic efficiency (kcat/Km) of the mutant V437I screened out by this method was 1.88 times higher than wild type. Next, to improve the growth of the engineered strain BLK07, the genes encoding for Xpk and Fbp were integrated into its genome to construct non-oxidative glycolysis (NOG) pathway. Finally, the CRISPR/Cas9 system was used to integrate the N6-pm1-V437I gene into the genome of BLK07. Pyruvic acid titer of the plasmid-free strain reached 42.20 g/L with an L-alanine conversion rate of 77.62% and a D-alanine resolution of 82.4%. This work would accelerate the industrial production of pyruvate and D-alanine by biocatalysis, and the HTS method established here could be used to screen other Pm1 mutants with high pyruvate titers.

Metabolic engineering for the synthesis of steviol glycosides: current status and future prospects.

With the pursuit of natural non-calorie sweeteners, steviol glycosides (SGs) have become one of the most popular natural sweeteners in the market. The SGs in Stevia are a mixture of SGs synthesized from steviol (a terpenoid). SGs are diterpenoids. Different SGs depend on the number and position of sugar groups on the core steviol backbone. This diversity comes from the processing of glycoside steviol by various glycosyltransferases. Due to the differences in glycosylation, each SG has unique sensory properties. At present, it is more complicated to extract high-quality SGs from plants, so the excavation of the metabolic pathways of engineered microorganisms to synthesize SGs has been extensively studied. Specifically, the expression of different glycosyltransferases in microbes is key to the synthesis of various SGs by engineered microorganisms. To trigger more researches on the functional characterization of the enzymes encoded by these genes, this review describes the latest research progresses of the related enzymes involved in SG biosynthesis and metabolic engineering.Key points• Outlines the research progress of key enzymes in the biosynthetic pathway of SGs• Factors affecting the catalytic capacity of stevia glucosyltransferase• Provide guidance for the efficient synthesis of SGs in microbial cell factories.

Assembly of pathway enzymes by engineering functional membrane microdomain components for improved N-acetylglucosamine synthesis in Bacillus subtilis.

Enzyme clustering can improve catalytic efficiency by facilitating the processing of intermediates. Functional membrane microdomains (FMMs) in bacteria can provide a platform for enzyme clustering. However, the amount of FMMs at the cell basal level is still facing great challenges in multi-enzyme immobilization. Here, using the nutraceutical N-acetylglucosamine (GlcNAc) synthesis in Bacillus subtilis as a model, we engineered FMM components to improve the enzyme assembly in FMMs. First, by overexpression of the SPFH (stomatin-prohibitin-flotillin-HflC/K) domain and YisP protein, an enzyme involved in the synthesis of squalene-derived polyisoprenoid, the membrane order of cells was increased, as verified using di-4-ANEPPDHQ staining. Then, two heterologous enzymes, GlcNAc-6-phosphate N-acetyltransferase (GNA1) and haloacid dehalogenase-like phosphatases (YqaB), required for GlcNAc synthesis were assembled into FMMs, and the GlcNAc titer in flask was increased to 8.30 ± 0.57 g/L, which was almost three times that of the control strains. Notably, FMM component modification can maintain the OD600 in stationary phase and reduce cell lysis in the later stage of fermentation. These results reveal that the improved plasma membrane ordering achieved by the engineering FMM components could not only promote the enzyme assembly into FMMs, but also improve the cell fitness.

Synthetic metabolic channel by functional membrane microdomains for compartmentalized flux control.

The anchoring of metabolic pathway enzymes to spatial scaffolds can significantly improve their reaction efficiency. Here, we successfully constructed a multi-enzyme complex assembly system able to enhance bioproduction in bacteria by using the endogenous spatial scaffolds─functional membrane microdomains (FMMs). First, using VA-TIRFM and SPT analysis, we reveal that FMMs possess high temporal and spatial stability at the plasma membrane and can be used as endogenous spatial scaffolds to organize enzyme pathways. Then, taking the synthesis of N-acetylglucosamine (GlcNAc) in Bacillus subtilis as a proof-of-concept demonstration, we found that anchoring of various enzymes required for GlcNAc synthesis onto FMMs to obtain the FMMs-multi-enzyme complex system resulted in a significant increase in GlcNAc titer and an effectively alleviate in cell lysis at the later stage of fermentation compared to that in control strains expressing the related enzymes in the cytoplasm. Combining with metabolic model and kinetics analysis, the existence of a constructed substrate channel that maximizes the reaction efficiency is verified. In summary, we propose a novel metabolic pathway assembly model which allowed improved titers and compartmentalized flux control with high spatial resolution in bacterial metabolism.