Yanyan Gu

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.

Metabolic engineering of Bacillus amyloliquefaciens for poly‐gamma‐glutamic acid (γ‐PGA) overproduction

We constructed a metabolically engineered glutamate‐independent Bacillus amyloliquefaciens strain with considerable γ‐PGA production. It was carried out by double‐deletion of the cwlO gene and epsA‐O cluster, as well as insertion of the vgb gene in the bacteria chromosome. The final generated strain NK‐PV elicited the highest production of γ‐PGA (5.12 g l−1), which was 63.2% higher than that of the wild‐type NK‐1 strain (3.14 g l−1). The γ‐PGA purity also improved in the NK‐PV strain of 80.4% compared with 76.8% for the control. Experiments on bacterial biofilm formation experiment showed that NK‐1 and NK‐c (ΔcwlO) strains can form biofilm; the epsA‐O deletion NK‐7 and NK‐PV strains could only form an incomplete biofilm.

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.

Cloning of ε-poly-L-lysine (ε-PL) synthetase gene from a newly isolated ε-PL-producing Streptomyces albulus NK660 and its heterologous expression in Streptomyces lividans.

ε‐Poly‐L‐lysine (ε‐PL), showing a wide range of antimicrobial activity, is now industrially produced as a food additive by a fermentation process. A new strain capable of producing ε‐PL was isolated from a soil sample collected from Gutian, Fujian Province, China. Based on its morphological and biochemical features and phylogenetic similarity with 16S rRNA gene, the strain was identified as Streptomyces albulus and named NK660. The yield of ε‐PL in 30 l fed‐batch fermentation with pH control was 4.2 g l−1 when using glycerol as the carbon source. The structure of ε‐PL was determined by nuclear magnetic resonance (NMR) and matrix‐assisted laser desorption/ionization–time of flight mass spectrometry (MALDI‐TOF MS). Previous studies have shown that the antimicrobial activity of ε‐PL is dependent on its molecular size. In this study, the polymerization degree of the ε‐PL produced by strain NK660 ranged from 19 to 33 L‐lysine monomers, with the main component consisting of 24–30 L‐lysine monomers, which implied that the ε‐PL might have higher antimicrobial activity. Furthermore, the ε‐PL synthetase gene (pls) was cloned from strain NK660 by genome walking. The pls gene with its native promoter was heterologously expressed in Streptomyces lividans ZX7, and the recombinant strain was capable of synthesizing ε‐PL. Here, we demonstrated for the first time heterologous expression of the pls gene in S. lividans. The heterologous expression of pls gene in S. lividans will open new avenues for elucidating the molecular mechanisms of ε‐PL synthesis.

Treatment of high-salinity chemical wastewater by indigenous bacteria – bioaugmented contact oxidation

A 90 m(3) biological contact oxidation system in chemical factory was bioaugmented with three strains of indigenous salt-tolerant bacteria. These three strains were screened from contaminative soil in situ. Their activity of growth and degradation was investigated with lab-scale experiments. Their salt-tolerant mechanism was confirmed to be compatible-solutes strategy for moderately halophilic bacteria, with amino acid and betaine playing important roles. The running conditions of the system were recorded for 150 days. The indigenous bacteria had such high suitability that the reactor got steady rapidly and the removal of COD maintained above 90%. It was introduced that biofilm fragments in sedimentation tank were inversely flowed to each reaction tank, and quantitative PCR demonstrated that this process could successfully maintain the bacterial abundance in the reaction tanks. In addition, the T-RFLP revealed that bioaugmented strains dominated over others in the biofilm.

Construction of a Bacillus amyloliquefaciens strain for high purity levan production.

Bacillus amyloliquefaciens NK-1 has the potential to produce levan and poly-gamma-glutamic acid (γ-PGA) simultaneously. However, it is not possible to purify each single product from the same strain because the extraction process is identical. We deleted the pgs cluster (for γ-PGA synthesis) from the NK-1 strain and constructed a γ-PGA-deficient NK-ΔLP strain. Nuclear magnetic results showed that the NK-ΔLP strain could produce high purity levan product. However, its levan titer was only 1.96 g L(-1) in the basal medium. Single-factor experimental and response surface methodology was used to optimize the culture condition, leading to levan titer of 13.9 and 22.6 g L(-1) in flask culture and in a 5-L bioreactor, respectively. The levan purity can reach to 92.7% after 48 h cultivation. Furthermore, the relationship between levanase (LevB) and levan molecular weight was studied. The results showed that LevB resulted in the production of low molecular weight levan and its expression level determined the ratio of high and low molecular weight levan. We also deleted the sac cluster (for levan synthesis) from the NK-1 strain and constructed a levan-deficient NK-L strain. The NK-L strain exhibited increased purity of γ-PGA product from 79.5 to 91.2%.

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

BackgroundSucrose 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.ResultsIn 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.ConclusionsTo 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.

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.

Genome Sequence of the ε-Poly-l-Lysine-Producing Strain Streptomyces albulus NK660, Isolated from Soil in Gutian, Fujian Province, China

ABSTRACT We determined the complete genome sequence of a soil bacterium, Streptomyces albulus NK660. It can produce ε-poly-l-lysine, which has antimicrobial activity against a spectrum of microorganisms. The genome of S. albulus NK660 contains a 9,360,281-bp linear chromosome and a 12,120-bp linear plasmid.

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

BackgroundPoly-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.ResultsIn 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.ConclusionsWe 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.