Synthesis of Highly Photoluminescent All-Inorganic CsPbX3 Nanocrystals via Interfacial Anion Exchange Reactions.

All-inorganic cesium lead halide perovskite CsPbX3 (X = Cl, Br, I) nanocrystals (NCs) have attracted significant attention owing to their fascinating electronic and optical properties. However, researchers still face challenges to achieve highly stable and photoluminescent CsPbX3 NCs at room temperature by the direct-synthesis method. Herein, we synthesize CsPbX3 NCs by a facile and environmentally friendly method, which uses an aqueous solution of metal halides to react with Cs4PbBr6 NCs via interfacial anion exchange reactions and without applying any pretreatment. This method produces monodisperse and air-stable CsPbX3 NCs with tunable spectra covering the entire visible range, narrow photoluminescence emission bandwidth, and high photoluminescence quantum yield (PL QY, 80%). In addition, the chemical transformation mechanism between Cs4PbBr6 NCs and CsPbX3 NCs was investigated. The Cs4PbBr6 NCs were converted to CsPbBr3 NCs first by stripping CsBr, and then, the as-prepared CsPbBr3 NCs reacted with metal halides to form CsPbX3 NCs. This work takes advantage of the chemical transformation mechanism of Cs4PbBr6 NCs and provides an efficient and environmentally friendly way to synthesize CsPbX3 NCs.

Investigating the transformation of CsPbBr3 nanocrystals into highly stable CsPbBr3/Cs4PbBr6 nanocrystals using ethyl acetate in a microchannel reactor.

The nanocrystals (NCs) of inorganic perovskites CsPbX3 and Cs4PbX6 (X = Cl, Br, I) are showing a great development potential due to their versatility of crystal structure. Here, we used a microchannel reactor to synthesize both CsPbBr3 NCs (CsPbBr3 NCs) and Cs4PbBr6 NCs with embedded CsPbBr3 (CsPbBr3/Cs4PbBr6 NCs). Via speed control of the precursor, ligands around the surface of NCs were effectively regulated by ethyl acetate, allowing the transformation from CsPbBr3 NCs to CsPbBr3/Cs4PbBr6 NCs in a short time, an outstanding stability of NCs, and a better crosslinking between NCs and polymer for the application of LEDs. Without any protection, the CsPbBr3/Cs4PbBr6 NCs, with a production rate of 28 mg min-1, retain more than 90% of the PL intensity after 84 d. Finally, the CsPbBr3/Cs4PbBr6 NCs were used to produce an LED device, and a wide color gamut of 122.8% NTSC or 91.7% Rec 2020 was attained.

Genetic and metabolic engineering for microbial production of poly-γ-glutamic acid.

Poly-γ-glutamic acid (γ-PGA) is a natural biopolymer of glutamic acid. The repeating units of γ-PGA may be derived exclusively from d-glutamic acid, or l-glutamic acid, or both. The monomer units are linked by amide bonds between the α-amino group and the γ-carboxylic acid group. γ-PGA is biodegradable, edible and water-soluble. It has numerous existing and emerging applications in processing of foods, medicines and cosmetics. This review focuses on microbial production of γ-PGA via genetically and metabolically engineered recombinant bacteria. Strategies for improving production of γ-PGA include modification of its biosynthesis pathway, enhancing the production of its precursor (glutamic acid), and preventing loss of the precursor to competing byproducts. These and other strategies are discussed. Heterologous synthesis of γ-PGA in industrial bacterial hosts that do not naturally produce γ-PGA is discussed. Emerging trends and the challenges affecting the production of γ-PGA are reviewed.

CRISPR-Mediated Genome Editing and Gene Repression in Scheffersomyces stipitis.

Scheffersomyces stipitis, renowned for its native xylose-utilizing capacity, has recently demonstrated its potential in producing health-promoting shikimate pathway derivatives. However, its broader application is hampered by the low transformation efficiency and the lack of genetic engineering tools to enable sophisticated genomic manipulations. S. stipitis employs the predominant non-homologous end joining (NHEJ) mechanism for repairing DNA double-strand breaks (DSB), which is less desired due to its incompetence in achieving precise genome editing. Using CRISPR technology, here a ku70Δku80Δ deficient strain in which homologous recombination (HR)-based genome editing appeared dominant for the first time in S. stipitis is constructed. To build all essential tools for efficiently manipulating this highly promising nonconventional microbial host, the gene knockdown tool is also established, and repression efficiency is improved by incorporating a transcriptional repressor Mxi1 into the CRISPR-dCas9 platform. All these results are obtained with the improved transformation efficiency, which is 191-fold higher than that obtained with the traditional parameters used in yeast transformation. This work paves the way for advancing a new microbial chassis and provides a guideline for developing efficient CRISPR tools in other nonconventional yeasts.

Efficacy of Various Preservatives on Extending Shelf Life of Vacuum-Packaged Raw Pork during 4°C Storage.

Uncontrolled bacterial growth and metabolic activities are responsible for the short shelf life of raw pork. Culture-independent analysis by 16S ribosome cDNA could reveal viable bacteria in raw pork. This study investigated microbial growth and volatile organic compounds of raw pork supplemented with various preservatives. Vacuum-packaged raw pork was stored at 4°C, after soaking in solutions of potassium sorbate, ε-poly-l-lysine, kojic acid (KA), or sodium diacetate, individually. Spoilage of raw pork was monitored by determining pH and total volatile basic nitrogen, whereas bacterial growth was determined by culture-dependent and culture-independent analyses. Data indicated that all the preservatives were able to inhibit bacterial growth and extend the shelf life of pork. High-throughput sequencing of 16S ribosome cDNA indicated that Pseudomonas was inhibited under vacuum conditions, whereas facultative anaerobes ( Acinetobacter, Photobacterium, Brochothrix, and Myroides) were the most active genera in the spoiled pork. Photobacterium was further inhibited by each preservative. The inhibition of Acinetobacter, Photobacterium, and Myroides could be responsible for the extended shelf life of vacuum-packaged pork; they were effectively inhibited by KA, which also induced the longest shelf life. Moreover, 19 types of volatile organic compounds were detected. 3-Methylbutanol, 3-methylbutanol acetate, 2-butanone, toluene, benzeneacetaldehyde, dimethyl trisulfide, and acetoin were associated with spoilage. Furthermore, KA is a potential preservative in raw pork; because no phenol was detectable within 35 days, excessive intake of phenol induced by preservatives was avoided.

Regulation of bacteria population behaviors by AI-2 "consumer cells" and "supplier cells".

BACKGROUND: Autoinducer-2 (AI-2) is a universal signal molecule and enables an individual bacteria to communicate with each other and ultimately control behaviors of the population. Harnessing the character of AI-2, two kinds of AI-2 "controller cells" ("consumer cells" and "supplier cells") were designed to "reprogram" the behaviors of entire population. RESULTS: For the consumer cells, genes associated with the uptake and processing of AI-2, which includes LsrACDB, LsrFG, LsrK, were overexpressed in varying combinations. Four consumer cell strains were constructed: Escherichia coli MG1655 pLsrACDB (NK-C1), MG1655 pLsrACDBK (NK-C2), MG1655 pLsrACDBFG (NK-C3) and MG1655 pLsrACDBFGK (NK-C4). The key enzymes responsible for production of AI-2, LuxS and Mtn, were also overexpressed, yielding strains MG1655 pLuxS (NK-SU1), and MG1655 pLuxS-Mtn (NK-SU2). All the consumer cells could decrease the environmental AI-2 concentration. NK-C2 and NK-C4 were most effective in AI-2 uptake and inhibited biofilm formation. While suppliers can increase the environmental AI-2 concentration and NK-SU2 was most effective in supplying AI-2 and facilitated biofilm formation. Further, reporter strain, MG1655 pLGFP was constructed. The expression of green fluorescent protein (GFP) in reporter cells was initiated and guided by AI-2. Mixture of consumer cells and reporter cells suggest that consumer cells can decrease the AI-2 concentration. And the supplier cells were co-cultured with reporter cells, indicating that supplier cells can provide more AI-2 compared to the control. CONCLUSIONS: The consumer cells and supplier cells could be used to regulate environmental AI-2 concentration and the biofilm formation. They can also modulate the AI-2 concentration when they were co-cultured with reporter cells. It can be envisioned that this system will become useful tools in synthetic biology and researching new antimicrobials.

Combinatorial metabolic engineering of Pseudomonas putida KT2440 for efficient mineralization of 1,2,3-trichloropropane.

An industrial waste, 1,2,3-trichloropropane (TCP), is toxic and extremely recalcitrant to biodegradation. To date, no natural TCP degraders able to mineralize TCP aerobically have been isolated. In this work, we engineered a biosafety Pseudomonas putida strain KT2440 for aerobic mineralization of TCP by implantation of a synthetic biodegradation pathway into the chromosome and further improved TCP mineralization using combinatorial engineering strategies. Initially, a synthetic pathway composed of haloalkane dehalogenase, haloalcohol dehalogenase and epoxide hydrolase was functionally assembled for the conversion of TCP into glycerol in P. putida KT2440. Then, the growth lag-phase of using glycerol as a growth precursor was eliminated by deleting the glpR gene, significantly enhancing the flux of carbon through the pathway. Subsequently, we improved the oxygen sequestering capacity of this strain through the heterologous expression of Vitreoscilla hemoglobin, which makes this strain able to mineralize TCP under oxygen-limited conditions. Lastly, we further improved intracellular energy charge (ATP/ADP ratio) and reducing power (NADPH/NADP+ ratio) by deleting flagella-related genes in the genome of P. putida KT2440. The resulting strain (named KTU-TGVF) could efficiently utilize TCP as the sole source of carbon for growth. Degradation studies in a bioreactor highlight the value of this engineered strain for TCP bioremediation.

Construction of energy-conserving sucrose utilization pathways for improving poly-γ-glutamic acid production in Bacillus amyloliquefaciens.

BACKGROUND: Sucrose is an naturally abundant and easily fermentable feedstock for various biochemical production processes. By now, several sucrose utilization pathways have been identified and characterized. Among them, the pathway consists of sucrose permease and sucrose phosphorylase is an energy-conserving sucrose utilization pathway because it consumes less ATP when comparing to other known pathways. Bacillus amyloliquefaciens NK-1 strain can use sucrose as the feedstock to produce poly-γ-glutamic acid (γ-PGA), a highly valuable biopolymer. The native sucrose utilization pathway in NK-1 strain consists of phosphoenolpyruvate-dependent phosphotransferase system and sucrose-6-P hydrolase and consumes more ATP than the energy-conserving sucrose utilization pathway. RESULTS: In this study, the native sucrose utilization pathway in NK-1 was firstly deleted and generated the B. amyloliquefaciens 3Δ strain. Then four combination of heterologous energy-conserving sucrose utilization pathways were constructed and introduced into the 3Δ strain. Results demonstrated that the combination of cscB (encodes sucrose permease) from Escherichia coli and sucP (encodes sucrose phosphorylase) from Bifidobacterium adolescentis showed the highest sucrose metabolic efficiency. The corresponding mutant consumed 49.4% more sucrose and produced 38.5% more γ-PGA than the NK-1 strain under the same fermentation conditions. CONCLUSIONS: To our best knowledge, this is the first report concerning the enhancement of the target product production by introducing the heterologous energy-conserving sucrose utilization pathways. Such a strategy can be easily extended to other microorganism hosts for reinforced biochemical production using sucrose as substrate.

Enhancing poly-γ-glutamic acid production in Bacillus amyloliquefaciens by introducing the glutamate synthesis features from Corynebacterium glutamicum.

BACKGROUND: Poly-γ-glutamic acid (γ-PGA) is a valuable polymer with glutamate as its sole precursor. Enhancement of the intracellular glutamate synthesis is a very important strategy for the improvement of γ-PGA production, especially for those glutamate-independent γ-PGA producing strains. Corynebacterium glutamicum has long been used for industrial glutamate production and it exhibits some unique features for glutamate synthesis; therefore introduction of these metabolic characters into the γ-PGA producing strain might lead to increased intracellular glutamate availability, and thus ultimate γ-PGA production. RESULTS: In this study, the unique glutamate synthesis features from C. glutamicum was introduced into the glutamate-independent γ-PGA producing Bacillus amyloliquefaciens NK-1 strain. After introducing the energy-saving NADPH-dependent glutamate dehydrogenase (NADPH-GDH) pathway, the NK-1 (pHT315-gdh) strain showed slightly increase (by 9.1%) in γ-PGA production. Moreover, an optimized metabolic toggle switch for controlling the expression of ɑ-oxoglutarate dehydrogenase complex (ODHC) was introduced into the NK-1 strain, because it was previously shown that the ODHC in C. glutamicum was completely inhibited when glutamate was actively produced. The obtained NK-PO1 (pHT01-xylR) strain showed 66.2% higher γ-PGA production than the NK-1 strain. However, the further combination of these two strategies (introducing both NADPH-GDH pathway and the metabolic toggle switch) did not lead to further increase of γ-PGA production but rather the resultant γ-PGA production was even lower than that in the NK-1 strain. CONCLUSIONS: We proposed new metabolic engineering strategies to improve the γ-PGA production in B. amyloliquefaciens. The NK-1 (pHT315-gdh) strain with the introduction of NADPH-GDH pathway showed 9.1% improvement in γ-PGA production. The NK-PO1 (pHT01-xylR) strain with the introduction of a metabolic toggle switch for controlling the expression of ODHC showed 66.2% higher γ-PGA production than the NK-1 strain. This work proposed a new strategy for improving the target product in microbial cell factories.

Elucidation of major contributors involved in nitrogen removal and transcription level of nitrogen-cycling genes in activated sludge from WWTPs.

We investigated nitrogen-cycle bacterial communities in activated sludge from 8 municipal wastewater treatment plants (WWTPs). Redundancy analyses (RDA) showed that temperature was the most significant driving force in shaping microbial community structure, followed by influent NH4+ and total nitrogen (TN). The diversity of ammonia oxidizing and nitrite reducing bacteria were investigated by the construction of amoA, nirS and nirK gene clone libraries. Phylogenetic analysis indicated that Thauera and Mesorhizobium were the predominant nitrite reducing bacteria, and Nitrosomonas was the only detected ammonia oxidizing bacteria in all samples. Quantification of transcription level of nirS and nirK genes indicated that nirS-type nitrite reducing bacteria played the dominant roles in nitrite reduction process. Transcription level of nirS gene positively correlated with influent NH4+ and TN significantly, whereas inversely linked with hydraulic retention time. Temperature had a strong positive correlation to transcription level of amoA gene. Overall, this study deepened our understanding of the major types of ammonia oxidizing and nitrite reducing bacteria in activated sludge of municipal WWTPs. The relationship between transcription level of nitrogen-cycle genes and operational or environmental variables of WWTPs revealed in this work could provide guidance for optimization of operating parameters and improving the performance of nitrogen removal.

Improvement of levan production in Bacillus amyloliquefaciens through metabolic optimization of regulatory elements.

Levan is a functional homopolymer of fructose with considerable applications in food, pharmaceutical and cosmetic industries. To improve the levan production in Bacillus amyloliquefaciens, the regulatory elements of sacB (encoding levansucrase) expression and levansucrase secretion were optimized. Four heterologous promoters were evaluated for sacB expression, and the Pgrac promoter led to the highest level for both sacB transcription and levansucrase enzyme activity. The levan production in the corresponding recombinant strain ΔLP-pHTPgrac reached 30.5 g/L, which was 114% higher than that of the control strain NK-ΔLP. In a further step, eight signal peptides were investigated (with Pgrac as the promoter for sacB expression) for their effects on the levansucrase secretion and levan production. The signal peptide yncM was identified as the optimal one, with a secretion efficiency of approximately 90%, and the levan production in the corresponding recombinant strain ΔLP-Y reached 37.4 g/L, which was 161% higher when compared with the control strains NK-ΔLP. Finally, fed-batch fermentation was carried out in 5-L bioreactors for levan production using the recombinant strain ΔLP-Y. A final levan concentration of 102 g/L was achieved, which is very close to the ever reported highest levan production level from the literature. To our best knowledge, this is the first report of the improvement of levan production through metabolic optimization for sacB expression and levansucrase secretion. The results from this study provided essential insights for systematically metabolic engineering of microbial cell factories for enhanced biochemical production.

Mutations in genes encoding antibiotic substances increase the synthesis of poly-γ-glutamic acid in Bacillus amyloliquefaciens LL3.

Poly-γ-glutamic acid (γ-PGA) is an important natural biopolymer that is used widely in fields of foods, medicine, cosmetics, and agriculture. Several B. amyloliquefaciens LL3 mutants were constructed to improve γ-PGA synthesis via single or multiple marker-less in-frame deletions of four gene clusters (itu, bae, srf, and fen) encoding antibiotic substances. γ-PGA synthesis by the Δsrf mutant showed a slight increase (4.1 g/L) compared with that of the wild-type strain (3.3 g/L). The ΔituΔsrf mutant showed increased γ-PGA yield from 3.3 to 4.5 g/L, with an increase of 36.4%. The γ-PGA yield of the ΔituΔsrfΔfen and ΔituΔsrfΔfenΔbae mutants did not show a further increase. The four gene clusters' roles in swarming motility and biofilm formation were also studied. The Δsrf and Δbae mutant strains were both significantly defective in swarming, indicating that bacillaene and surfactin are involved in swarming motility of B. amyloliquefaciens LL3. Furthermore, Δsrf and Δitu mutant strains were obviously defective in biofilm formation; therefore, iturin and surfactin must play important roles in biofilm formation in B. amyloliquefaciens LL3.

High-Throughput Sequencing of Viable Microbial Communities in Raw Pork Subjected to a Fast Cooling Process.

This study aimed to investigate the effect of the fast cooling process on the microbiological community in chilled fresh pork during storage. We established a culture-independent method to study viable microbes in raw pork. Tray-packaged fresh pork and chilled fresh pork were completely spoiled after 18 and 49 d in aseptic bags at 4 °C, respectively. 16S/18S ribosomal RNAs were reverse transcribed to cDNA to characterize the activity of viable bacteria/fungi in the 2 types of pork. Both cDNA and total DNA were analyzed by high-throughput sequencing, which revealed that viable Bacteroides sp. were the most active genus in rotten pork, although viable Myroides sp. and Pseudomonas sp. were also active. Moreover, viable fungi were only detected in chilled fresh pork. The sequencing results revealed that the fast cooling process could suppress the growth of microbes present initially in the raw meat to extend its shelf life. Our results also suggested that fungi associated with pork spoilage could not grow well in aseptic tray-packaged conditions.

Effects of MreB paralogs on poly-γ-glutamic acid synthesis and cell morphology in Bacillus amyloliquefaciens.

Actin-like MreB paralogs play important roles in cell shape maintenance, cell wall synthesis and the regulation of the D,L-endopeptidases, CwlO and LytE. The gram-positive bacteria, Bacillus amyloliquefaciens LL3, is a poly-γ-glutamic acid (γ-PGA) producing strain that contains three MreB paralogs: MreB, Mbl and MreBH. In B. amyloliquefaciens, CwlO and LytE can degrade γ-PGA. In this study, we aimed to test the hypothesis that modulating transcript levels of MreB paralogs would alter the synthesis and degradation of γ-PGA. The results showed that overexpression or inhibition of MreB, Mbl or MreBH had distinct effects on cell morphology and the molecular weight of the γ-PGA products. In fermentation medium, cells of mreB inhibition mutant were 50.2% longer than LL3, and the γ-PGA titer increased by 55.7%. However, changing the expression level of mbl showed only slight effects on the morphology, γ-PGA molecular weight and titer. In the mreBH inhibition mutant, γ-PGA production and its molecular weight increased by 56.7% and 19.4%, respectively. These results confirmed our hypothesis that suppressing the expression of MreB paralogs might reduce γ-PGA degradation, and that improving the cell size could strengthen γ-PGA synthesis. This is the first report of enhanced γ-PGA production via suppression of actin-like MreB paralogs.

Engineering Pseudomonas putida KT2440 for simultaneous degradation of carbofuran and chlorpyrifos.

Currently, chlorpyrifos (CP) and carbofuran are often applied together to control major agricultural pests in many developing countries, in most cases, they are simultaneously detected in agricultural soils. Some cost-effective techniques are required for the remediation of combined pollution caused by multiple pesticides. In this work, we aim at constructing a detectable recombinant microorganism with the capacity to simultaneously degrade CP and carbofuran. To achieve this purpose, CP/carbofuran hydrolase genes and gfp were integrated into the chromosome of a biosafety strain Pseudomonas putida KT2440 using a chromosomal scarless modification strategy with upp as a counter-selectable marker. The toxicity of the hydrolysis products was significantly lower compared with the parent compounds. The recombinant strain could utilize CP or carbofuran as the sole source of carbon for growth. The inoculation of the recombinant strain to soils treated with carbofuran and CP resulted in a higher degradation rate than in noninoculated soils. Introduced green fluorescent protein can be employed as a biomarker to track the recombinant strain during bioremediation. Therefore, the recombinant strain has potential to be applied for in situ bioremediation of soil co-contaminated with carbofuran and CP.

Metabolic Engineering of Pseudomonas putida KT2440 for Complete Mineralization of Methyl Parathion and γ-Hexachlorocyclohexane.

Agricultural soils are often cocontaminated with multiple pesticides. Unfortunately, microorganisms isolated from natural environments do not possess the ability to simultaneously degrade different classes of pesticides. Currently, we can use the approaches of synthetic biology to create a strain endowed with various catabolic pathways that do not exist in a natural microorganism. Here, we describe the metabolic engineering of a biosafety Pseudomonas putida strain KT2440 for complete mineralization of methyl parathion (MP) and γ-hexachlorocyclohexane (γ-HCH) by functional assembly of the MP and γ-HCH mineralization pathways. The engineered strain was genetically stable, and no growth inhibition was observed. Such a strain not only would reduce the toxicity of MP and γ-HCH but also would prevent the accumulation of potentially toxic intermediates in the environment. Furthermore, expression of Vitreoscilla hemoglobin improved the ability of the engineered strain to sequester O2. The inoculation of the engineered strain to soils treated with MP and γ-HCH resulted in a higher degradation rate than in noninoculated soils. Moreover, introduced GFP may be used to monitor the activity of the engineered strain during bioremediation. The engineered strain may be a promising candidate for in situ bioremediation of soil cocontaminated with MP and γ-HCH.

Effects of Chromosomal Integration of the Vitreoscilla Hemoglobin Gene (vgb) and S-Adenosylmethionine Synthetase Gene (metK) on ε-Poly-L-Lysine Synthesis in Streptomyces albulus NK660.

ε-Poly-L-lysine (ε-PL) is a widely used natural food preservative. To test the effects of the Vitreoscilla hemoglobin (VHb) and S-adenosylmethionine (SAM) on ε-PL synthesis in Streptomyces albulus NK660, the heterologous VHb gene (vgb) and SAM synthetase gene (metK) were inserted into the S. albulus NK660 chromosome under the control of the constitutive ermE* promoter. CO-difference spectrum analysis showed S. albulus NK660-VHb strain could express functional VHb. S. albulus NK660-VHb produced 26.67 % higher ε-PL and 14.57 % higher biomass than the wild-type control, respectively. Reversed-phase high-pressure liquid chromatography (RP-HPLC) results showed the overexpression of the metK gene resulted in increased intracellular SAM synthesis in S. albulus NK660-SAM, which caused increases of biomass as well as the transcription level of ε-PL synthetase gene (pls). Results indicated that the expression of vgb and metK gene improved on ε-PL synthesis and biomass for S. albulus NK660, respectively.

Enhancement of medium-chain-length polyhydroxyalkanoates biosynthesis from glucose by metabolic engineering in Pseudomonas mendocina.

OBJECTIVES: To enhance the biosynthesis of medium-chain-length polyhydroxyalkanoates (PHAMCL) from glucose in Pseudomonas mendocina NK-01, metabolic engineering strategies were used to block or enhance related pathways. RESULTS: Pseudomonas mendocina NK-01 produces PHAMCL from glucose. Besides the alginate oligosaccharide biosynthetic pathway proved by our previous study, UDP-D-glucose and dTDP-L-rhamnose biosynthetic pathways were identified. These might compete for glucose with the PHAMCL biosynthesis. First, the alg operon, galU and rmlC gene were deleted one by one, resulting in NK-U-1(∆alg), NK-U-2 (∆alg∆galU), NK-U-3(alg∆galU∆rmlC). After fermentation for 36 h, the cell dry weight (CDW) and PHAMCL production of these strains were determined. Compared with NK-U: 1) NK-U-1 produced elevated CDW (from 3.19 ± 0.16 to 3.5 ± 0.11 g/l) and equal PHAMCL (from 0.78 ± 0.06 to 0.79 ± 0.07 g/l); 2) NK-U-2 produced more CDW (from 3.19 ± 0.16 to 3.55 ± 0.23 g/l) and PHAMCL (from 0.78 ± 0.06 to 1.05 ± 0.07 g/l); 3) CDW and PHAMCL dramatically decreased in NK-U-3 (1.53 ± 0.21 and 0.41 ± 0.09 g/l, respectively). Additionally, the phaG gene was overexpressed in strain NK-U-2. Although CDW of NK-U-2/phaG decreased to 1.29 ± 0.2 g/l, PHA titer (%CDW) significantly increased from 24.5 % up to 51.2 %. CONCLUSION: The PHAMCL biosynthetic pathway was enhanced by blocking branched metabolic pathways in combination with overexpressing phaG gene.

Recruiting a new strategy to improve levan production in Bacillus amyloliquefaciens.

Microbial levan is an important biopolymer with considerable potential in food and medical applications. Bacillus amyloliquefaciens NK-ΔLP strain can produce high-purity, low-molecular-weight levan, but production is relatively low. To enhance the production of levan, six extracellular protease genes (bpr, epr, mpr, vpr, nprE and aprE), together with the tasA gene (encoding the major biofilm matrix protein TasA) and the pgsBCA cluster (responsible for poly-γ-glutamic acid (γ-PGA) synthesis), were intentionally knocked out in the Bacillus amyloliquefaciens NK-1 strain. The highest levan production (31.1 g/L) was obtained from the NK-Q-7 strain (ΔtasA, Δbpr, Δepr, Δmpr, Δvpr, ΔnprE, ΔaprE and ΔpgsBCA), which was 103% higher than that of the NK-ΔLP strain (ΔpgsBCA) (15.3 g/L). Furthermore, the NK-Q-7 strain also showed a 94.1% increase in α-amylase production compared with NK-ΔLP strain, suggesting a positive effect of extracellular protease genes deficient on the production of endogenously secreted proteins. This is the first report of the improvement of levan production in microbes deficient in extracellular proteases and TasA, and the NK-Q-7 strain exhibits outstanding characteristics for extracellular protein production or extracellular protein related product synthesis.

Improved poly-γ-glutamic acid production in Bacillus amyloliquefaciens by modular pathway engineering.

A Bacillus amyloliquefaciens strain with enhanced γ-PGA production was constructed by metabolically engineering its γ-PGA synthesis-related metabolic networks: by-products synthesis, γ-PGA degradation, glutamate precursor synthesis, γ-PGA synthesis and autoinducer synthesis. The genes involved in by-products synthesis were firstly deleted from the starting NK-1 strain. The obtained NK-E7 strain with deletions of the epsA-O (responsible for extracellular polysaccharide synthesis), sac (responsible for levan synthesis), lps (responsible for lipopolysaccharide synthesis) and pta (encoding phosphotransacetylase) genes, showed increased γ-PGA purity and slight increase of γ-PGA titer from 3.8 to 4.15 g/L. The γ-PGA degrading genes pgdS (encoding poly-gamma-glutamate depolymerase) and cwlO (encoding cell wall hydrolase) were further deleted. The obtained NK-E10 strain showed further increased γ-PGA production from 4.15 to 9.18 g/L. The autoinducer AI-2 synthetase gene luxS was deleted in NK-E10 strain and the resulting NK-E11 strain showed comparable γ-PGA titer to NK-E10 (from 9.18 to 9.54 g/L). In addition, we overexpressed the pgsBCA genes (encoding γ-PGA synthetase) in NK-E11 strain; however, the overexpression of these genes led to a decrease in γ-PGA production. Finally, the rocG gene (encoding glutamate dehydrogenase) and the glnA gene (glutamine synthetase) were repressed by the expression of synthetic small regulatory RNAs in NK-E11 strain. The rocG-repressed NK-anti-rocG strain exhibited the highest γ-PGA titer (11.04 g/L), which was 2.91-fold higher than that of the NK-1 strain. Fed-batch cultivation of the NK-anti-rocG strain resulted in a final γ-PGA titer of 20.3g/L, which was 5.34-fold higher than that of the NK-1 strain in shaking flasks. This work is the first report of a systematically metabolic engineering approach that significantly enhanced γ-PGA production in a B. amyloliquefaciens strain. The engineering strategies explored here are also useful for engineering cell factories for the production of γ-PGA or of other valuable metabolites.