Construction of a poly-γ-glutamic acid production improved Bacillus amyloliquefaciens strain

Poly-γ-glutamic acid (γ-PGA) is an important, naturally occurring polyamide consisting of D/L-glutamate monomers. Unlike typical peptide linkages, the amide linkages in γ-PGA are formed between the α-amino group and the γ-carboxyl group. γ-PGA exhibits many favorable features such as biodegradable, water soluble, edible and non-toxic to humans and the environment. Therefore, it has been widely used in fields of foods, medicines, cosmetics and agriculture and many unique applications, such as a sustained release material and drug carrier, curable biological adhesive, biodegradable fibres, and highly water absorbable hydrogels.

γ-PGA-producing strains are classified as either glutamate-dependent strains or glutamate-independent strains. Glutamate-independent strains are preferable for industrial production because of their low cost and simplified fermentation process. However, the majority of the glutamate-independent strains produce less γ-PGA than the glutamate-dependent strains. Therefore, the construction of a glutamate-independent strain with high γ-PGA yield is important for industrial applications.

In this project, we used modular pathway engineering to simultaneously optimize the entire biosynthesis pathways, and fine-tune the synthetic pathways and balance the metabolism in the glutamate-independent B. amyloliquefaciens NK-1 strain. A schematic of this engineering approach is shown in Fig. 1.

Fig. 1 Schematic of modular engineering approach in Bacillus amyloliquefaciens NK-1 strain. The red marks indicate the genes modified in the optimized pathway.

We aimed to improve γ-PGA production by carrying out the following five tasks: (1) block the pathways for by-product synthesis by knocking out four genes associated with bacterial polysaccharides: the eps cluster, the sac cluster, glyc (responsible for glycogen synthesis) and lps, as well as two genes that are associated with the two micromolecular products lactate and acetate, ldh and pta; (2) delete the genes pgdS and cwlO conding for γ-PGA-degrading enzymes; (3) delete the cellular autoinducer AI-2 synthetase gene luxS to make the strain tolerate environmental stress; (4) overexpress the pgsBCA genes by inserting a P43 promoter upstream of the cluster; (5) use synthetic small RNAs (sRNAs) to repress the expression of rocG and glnA genes to increase the amount of intracellular glutamate.

To obtain high purity and production of γ-PGA product, four bacterial polysaccharides associate genes: the eps cluster, sac cluster, glyc gene and lps gene were firstly deleted in the NK-1 strain in sequence and the constructed strains were designated as NK-E1, NK-E2, NK-E3 and NK-E4. The fermentation results were shown in Table 1. Contrast to our prediction, ploysacchride genes deletion did not significant affect the γ-PGA production and only the NK-E1 strain showed a silightly increase in γ-PGA production. Although the gene deletions had little effects on γ-PGA production, the purity of γ-PGA products significantly increased. γ-PGA product from the NK-E5 strian exhibited the highest purity of 95.2%, which was significantly higher than that of the NK-1 strain (78.6%).

Table 1 Comparison of γ-PGA fermentation results between NK-1 and mutant strains

Strains

γ-PGA titer (g/L)

Molecular Weight

(KDa)

Viscosity (cP)

Purity (%)

NK-1

3.8±0.11

428.6±27.8

34.4±2.5

78.6±4.1

NK-E1

3.94±0.09

414.1±27.0

62.3±4.5

83.2±5.3

NK-E2

3.25±0.03

377.5±24.4

123.5±16.8

91.5±2.1

NK-E3

2.64±0.31

380.9±17.7

142.3±11.2

94.4±0.6

NK-E4

2.96±0.47

376.5±24.3

107.2±39.3

93.9±1.1

NK-E5

3.62±0.18

391.2±27.9

268.7±57.0

95.2±0.9


Our previous work indicates that the NK-1 strain can produce small molecular by-products such as: lactate, acetate, ethanol and butanediol, which will distribute a large amount of carbon source and energy used for target product synthesis. Furthermore, the accumulation of acidic small molecules of lactate and acetate is also toxic to cell growth. To improve γ-PGA production, we blocked the pathways for lactate and acetate synthesis. The ldh gene and pta gene were single deleted and double deleted in the NK-E5 strain and constructed the NK-E6 (Δldh), NK-E7 (Δpta) and NK-E8 (Δldh and Δpta) strains. The fermentation results were shown in Fig. 2. Although the defect of acetate synthesis slightly inhibited the cell growth, the NK-E7 strain showed a 11% improvement in γ-PGA production (Fig. 2a, b) and the γ-PGA purity was 94.8%. The production of actate in these strains were also determined (Fig. 2c). The NK-E5 strain and NK-E6 strain could produce about 2.26 g/L and 1.58 g/L acetate, respectively; and the pta gene deletion NK-E7 and NK-E8 strains showed significant decrease in acetate production. However, trace amount of acetate could still be detected at end stage of fermentation. Some alternative pathways relate to acetate production contribute to its synthesis such as the aldehyde dehydrogenase can catalyze acetaldehyde to the acetate.

The ldh gene deletion NK-E6 and NK-E8 strains showed significant decrease in cell growth compared with NK-E5 strain (Fig. 2a); and the γ-PGA production was also significantly decreased about 81% and 40% compared with NK-E5 strain, respectively (Fig. 2b). The inactivation of LDH probably leads to the disruption of NADH/NAD+ redox balance thereafter inhibits cells growth. As shown in Fig. 2d, the ldh gene deletion strains could still produce lactate, which might related to existence of the alternative pathway for lactate synthesis.

Fig. 2 Fermentation results from NK-E5, NK-E6, NK-E7 and NK-E8 strains. (A) Time curves of cell growth of NK-E5 and the mutant strains. (B) γ-PGA fermentation results of NK-E5 and the mutant strains. Acetic acid (C) and lactic acid (D) production in NK-E5 and the mutant strains. Values represent means ± SD.


In our previous work, the effects of γ-PGA degrading genes deletions on its production was investigated in NK-1 strain and found that the pgdS and cwlO genes double deletion NK-pc strain showed the highest 93 % increase in γ-PGA production. Thus, we determined to delete the two genes in the NK-E7 strain. The cwlO gene deletion NK-E9 strain showed 39.8 % increased in γ-PGA titer (5.8 g/L) compared with the NK-E7 strain (4.15 g/L). The cwlO and pgdS double deletion NK-E10 strain showed 52.3 % increase in γ-PGA titer (6.32 g/L) compared with the NK-E7 strain (Table 2).

Although increased, γ-PGA production in NK-E10 strain was still not that high. As the increase of γ-PGA production and purity will affect the pH condition of the fermentation broth, we supposed that the low pH condition might be harmful to cell thereafter inhibited the γ-PGA production. To verify our speculation, we measured the pH values of strains NK-1, NK-E5, NK-E7, NK-E9 and NK-E10. As shown in Table 2, the pH value indeed decreased with the increase of γ-PGA production. To deal with this problem, we optimized the ratio of K2HPO4/KH2PO4 to obtain a more appropriate buffer system. The results showed that when the ratio of K2HPO4/KH2PO4 was 160 mM/120 mM, the NK-E10 strain exhibited the highest γ-PGA titer (9.18 g/L), which was 45.3% higher than that in previous γ-PGA fermentation medium. The final pH value of NK-E10 in the optimized fermentation broth (P5 medium) was 6.1, which was higher than that in the γ-PGA fermentation medium (5.49). These results indicated that low pH in the NK-E10 fermentation broth could influence the increase of γ-PGA production and the optimized buffer system can improve the fermentation condition thereafter increase the γ-PGA production.

Table 2 γ-PGA production and pH value of the strains

Strains

γ-PGA titer (g/L)

pH

NK-1

3.69±0.13

5.96±0.04

NK-E5

3.64±0.20

5.86±0.04

NK-E7

4.15±0.05

5.77±0.03

NK-E9

5.80±0.40

5.55±0.01

NK-E10

6.32±0.32

5.49±0.01

NK-1 (P5)#

2.31±0.19

6.19±0.05

NK-E10 (P5)#

9.18±0.13

6.10±0.02

#Represents the fermentation results in the P5 medium

Scientists had deleted the luxS gene in E. coli YH19 strain and the generated strain could tolerate the stress caused by the increased fuel production and showed increased production of isobutanol. To determine the effects of AI-2 on γ-PGA production, the luxS gene was deleted in the NK-E10 strain. The resulting NK-E11 strain showed a 11% increase in dry cell weight and its γ-PGA titer also slightly increased from 9.18 g/L to 9.54 g/L comparable with the NK-E10 strain.

To further improve γ-PGA production, the pgsBCA genes were overexpressed by inserting a strong promoter P43 from B. subtilis 168 into the upstream of the pgs cluster and resulting the NK-E12 strain (Fig. 3). Western blot results showed that the PgsB expression level was also increased in the NK-E12 strain compared the control strain (Fig. 3). As the pgsB, pgsC, pgsA genes are controlled by the same promoter, thus we concluded that the γ-PGA synthetase (PgsBCA) was overexpressed in the NK-E12 strain. In contrast to our speculation, the increased expression of PgsBCA did not lead to the increase of γ-PGA production. The γ-PGA production decreased 15.6% compared to NK-E11 strain. Bacterial membrane is important for substance transportation, cell growth and other metabolic activity. The PgsBCA expression level is strictly regulated by the cell global performance. The increased amount of PgsBCA in NK-12 strain might disrupt the cell balance or other membrane-associated metabolic activity thereafter result in γ-PGA production decrease.

Fig. 3 The pgsBCA genes overexpression. Strategy for P43 promoter insertion (a); Western blot results of PgsB protein expression between NK-E11 strain and NK-E12 strain (b).

Glutamate is the only precursor for γ-PGA synthesis, blocking the pathways for glutamic acid usage seems to be a way for glutamate accumulation. Glutamate in vivo can be degraded by RocG and GudB to produce 2-oxoglutarate. RocG is enzymatically active and mostly conributes to the glutamate degradation. Glutamate can also be used to produce glutamine by GlnA for the cell to assimilate ammonium. As glutamate plays an important role in cell growth and it is also a most important intersection linking carbon to nirogen metabolism, thus it is not wise to directly delete the genes related for glutamate usage. The trans-action sRNA can be exploited for fine flux control. The sRNA related engineering strategy has many advantages. The most outstanding feature is that it can dynamically repress the target gene expression without shutting down it. Thus it can be used for the study of metabolic key genes. Therefore, we used synthetic sRNAs to repress the expression of rocG and glnA genes to decrease the competitive pathways of glutamate usage.

The plasmids with sRNAs were transported into the NK-E11 strain (Fig. 4) and the fermentation results were showed in Fig. 5. The GDH activities from NK-anti-rocG and NK-anti-glnA-rocG strains were repressed to 0.04 mU/mg and 0.10 mU/mg, which were about 6.1% and 14.3% of the control NK-E11 strain (Fig. 5a). The GS activities from NK-anti-glnA and NK-anti-glnA-rocG strains were repressed to 0.39 U/mg and 0.38 U/mg, which were about 74.5% and 72.6% of the control NK-E11 strain (Fig. 5b). As shown in Fig. 5c. The anti-glnA sRNA expression strains NK-anti-glnA and NK-anti-glnA-rocG showed γ-PGA titers decreased of 55.9% and 44.3% to 4.11g/L and 5.19 g/L, respectively, compared with the control NK-E11 strain (9.32 g/L). The anti-rocG sRNA expression strain NK-anti-rocG showed γ-PGA titer increased about 18.5% to 11.04 g/L compared with the control NK-E11 strain. These results indicated that the full function of GS was important for γ-PGA production.

To evaluate the performance of the γ-PGA overproducing strain NK-anti-rocG in a more stable and comfortable condition, fed-batch culture was carried out in a 5 L up-scaled system. As showed in Fig. 5d, the highest titer was obtained at 54 h and the NK-anti-rocG strain could produce 20.3 g/L γ-PGA, which was 5.34-fold higher than that obtained from NK-1 strain in flask.

Fig. 4 Synthetic sRNAs design and expression. (A) mechanism of translation repression by sRNA. (B) structures of anti-glnA sRNA (a) and anti-rocG sRNA (b). The synthetic sRNAs are composed of three parts: a target mRNA-binding sequence, a scaffold sequence and a transcriptional terminator sequence. (C) designs for synthetic sRNAs and hfq gene expression. The synthetic sRNAs were put under the promoters from B. subtilis 168 (PBSP25 and Pupp) and expressed by plasmid pWH1520.

The hfq gene was also co-expressed with the synthetic sRNAs under the promoter of Phbs.


Fig. 5 The GDH activities (A) and GS activities (B) in NK-E11and the mutant strains after 30 h cultivation in P5 medium. γ-PGA fermentation results of NK-E11 and the mutant strains (C). Time curves of process parameters in a 5 L fermenter of the NK-anti-rocG strain (D). Values represent means ± SD.

Although the γ-PGA production in NK-anti-rocG strain is significantly increased, it is still too low to meet the industrial demand. More works must be done to improve the γ-PGA production in the future.

Up to now, all the experiments have been finished and the results have been published on Metabolic Engineering (Top journal).




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