Promoting Cell Growth And Poly-Y-Glutamic Acid Production By Boosting The Synthesis of Alanine And D-Alanyl-D-Alanine In Bacillus Licheniformis

12 Objective The production of some bio-chemicals affected by the cell growth. This study aimed at 13 promoting the cell growth by overexpressing the synthesis of peptidoglycans tetrapeptide tail 14 components to improve poly-γ-glutamic acid (γ-PGA) production. 15 Results L-alanine, D-alanine and D-alanyl-D-alanine are primary precursors for the synthesis of 16 peptidoglycans tetrapeptide tail. The addition of L-alanine and D-alanine significantly increased both the 17 cell growth and production of γ-PGA. Then, several genes encoding key enzymes for L/D-alanine and 18 D-alanyl-D-alanine biosynthesis were overexpressed respectively, including ald (encoding alanine 19 dehydrogenase), dal (encoding alanine racemase) and ddl (encoding D-alanine ligase). The results 20 showed that the overexpression of genes ald, dal and ddl increased the production of γ-PGA by 19.72%, 21 15.91% and 60.90%, and increased the microbial biomass by 15.58%, 18.34% and 49.85%, respectively. 22 Moreover, we demonstrated that the overexpression of genes ald, dal and ddl increased γ-PGA 23 production mainly by enhancing cell growth rather than providing more precursors. 24 Conclusions This work illustrated the importance of the L/D-alanine and D-alanyl-D-alanine synthesis 25 to the cell growth and the high yield of γ-PGA, and provided an effective strategy for producing γ-PGA. 26


Introduction
Bacterial cell walls are characterized by the presence of peptidoglycans, macromolecules built 32 from sugars and peptides, which help to maintain cell shape and integrity and balance the intracellular 33 osmotic pressure (Brown et al. 2020). Bacterial cell wall synthesis is closely related to cell growth, and 34 disruption of proper bacterial cell wall formation makes the cell highly sensitive to common 35 environmental pressure, such as high salinity or antibiotics (Das et al. 2011). Thus, the methods of 36 engineering cell wall component were generally developed to improve cell integrity and production of 37 biochemicals. For instance, Son et al. suppressed cell lysis and increased squalene production by 38 approximately 12% through activating the cell wall integrity pathway (Son et al. 2020). In addition, 39 elevation of membrane cardiolipin biosynthesis and repression of the cell division initiator protein FtsZ 40 also increased the OD600 by 86% and increased the HA titer by 204% (Westbrook et al. 2018). Therefore, 41 engineering cell wall component or shape might be feasible strategies to increase metabolites production. oxygen and limited the absorption or utilization of nutrients during the fermentation, which property 53 hindered the cell growth and the synthesis of γ-PGA (Hsueh et al. 2017). Previous study in our lab found 54 that the decrease of negative charge on cell wall surface could significantly improve cell growth and γ-55 PGA production in B. licheniformis (He et al. 2019). So, cell wall properties are closely related to cell 56 growth and γ-PGA synthesis. In this study, alanine dehydrogenase (encode by ald), alanine racemase 57 (encode by dal), D-alanyl-D-alanine ligase (encode by ddl) were respectively overexpressed to explore 58 the effects of L/D-alanine and D-alanyl-D-alanine which associated with peptidoglycans tetrapeptide tail 59 synthesis on cell growth and γ-PGA production.  Table 1 67 Medium and cultivation conditions. LB medium (10 g/L Tryptone, 5 g/L yeast extract, 10 g/L 68 NaCl, pH 7.2) was served as the basic medium for the cultivation of B. licheniformis and E. coli, and 20 69 μg/mL kanamycin, 50 μg/mL ampicillin or 20 μg/mL tetracycline were added into the medium when 70 necessary. The seed culture of B. licheniformis was prepared in 250 mL flasks with 50 mL LB medium, 71 and incubated in the rotatory shaker with 180 rpm at 37ºC for 10-12 h until OD600 reached 4.0~4.5. Then, 72 the seeds were transferred into the γ-PGA production medium, consisting of (per liter) 80 g glucose, 10 g MnSO4·7H2O at pH 7.2. The fermentation was performed in the rotatory shaker with 220 rpm at 37ºC overexpression strain was used as an example and which referred to the previous method (Cai et al. 2018).

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Briefly, P43 promoter from B. subtilis 168, ald gene and amyL terminator from B. licheniformis WX-02 79 were amplified by the corresponding primers ( Table 2), fused by SOE-PCR, and the fused fragment was 80 inserted into pHY300 at the restriction sites Xba I and EcoR I. PCR verification and DNA sequence 81 confirmed that the ald expression vector was constructed successfully, named pHY-ald. Then, pHY-ald 82 was electro-transferred into B. licheniformis WX-02 to construct the ald overexpression strain, named 83 WX-02/pHY-ald. The strain harboring the empty pHY300PLK was used as the control. Engineered 84 strains WX-02/pHY-ald, WX-02/pHY-dal and WX-02/pHY-ddl were constructed with the same method. Table 2 86 Analytical methods

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Cell growth curve was determined by measuring the optical density at 600 nm (OD600) in LB 88 medium. γ-PGA titer and biomass were detected by the method described in the previous research (He

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The addition of L-alanine or D-alanine can promote the cell growth and γ-PGA production.

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In order to investigate the effects of L-alanine or D-alanine on cell growth, growth curves of B. 99 licheniformis WX-02 were measured upon addition of different concentrations of exogenous L-100 alanine or D-alanine to the LB media (Fig. 2). Firstly, the OD600 of WX-02 in LB medium with L-alanine 101 addition were detected ( Fig. 2A), which showed a significant increasement in cell growth when 0.1 g/L 102 L-alanine was added to the medium compared with the control group (no L-alanine addition). It was 103 worthy for us to note that the cell growth does not always improve with the concentration of added L-104 alanine increases. The OD600 of cells were even decreased when 0.2, 0.3 and 0.4 g/L of L-alanine were 105 added to the medium compared with that of 0.1 g/L. These results suggested that the addition of L-alanine 106 can boost bacterial cell growth, but the concentration of added L-alanine was need to be considered.

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Then, the effect of D-alanine on cell growth was explored in WX-02 (Fig. 2B). The addition of D-108 alanine in medium promoted the cell growth of WX-02, and the OD600 increased most with 0.1 g/L D-109 alanine addition. From the above results, it was found that an appropriate concentration of L/D-alanine 110 could promote the cell growth, and the high concentration of L/D-alanine could hinder cell growth.

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showed that the supplement of L-alanine and D-alanine could effectively improve γ-PGA production 115 (Fig. 3). The titer of γ-PGA was increased by 14.92% compared with the control (without L-alanine 116 addition) when 0.2 g/L L-alanine was added into the medium (Fig. 3A). In addition, the γ-PGA titer was ( Fig. 3B). Therefore, the results indicated that the addition of L-alanine and D-alanine with appropriate primary precursors for D-alanyl-D-alanine synthesis, and D-alanyl-D-alanine is subsequently involved 123 in the synthesis of tetrapeptide tails during peptidoglycan assembly (Fig. 1). In order to explore effects strains had no significant difference compared with the control strain (Fig. 4B), which further indicated 143 that γ-PGA synthesis was promoted by enhancing cell growth rather than affecting its precursors 144 supplement when ald, dal and ddl were overexpressed.  (Table 3). It showed us that the overexpression of genes ald, dal and 155 ddl leaded a significant decrease in the synthesis of by-products, which was more conducive for cell 156 growth and γ-PGA synthesis. Table 3 158 Conclusion 159 γ-PGA is a kind of multifunctional biopolymer with many applications, and its high viscosity