Construction of the AQ Synthesis Module
The E.coli strain p01/BW25113, overexpressing BacD from Bacillus subtilis (BsBacD) which catalyzes the formation of AQ from alanine and glutamine, was constructed for the production of AQ. Extracellular AQ concentration was measured by HPLC, and 2.0 mM AQ was obtained (Fig. 2).
In E. coli, peptidases encoded by pepA, pepB, pepD and pepN, have been reported to degrade a broad spectrum of dipeptides[20, 21], and inactivating them might reduce AQ degradation. It was reported that deletion of dpp, encoding a dipeptide ABC transporter increased AQ accumulation [20, 22, 39]. By knocking out the genes pepN, pepA, pepB, pepD and dpp, the degradation of AQ was alleviated. In the starting host BW25113, 20 mM AQ was completely degraded after 3 hours, compared to only 1.3 mM in the chassis AQ09 (BW25113ΔpepN, ΔpepA, ΔpepB, ΔpepD, Δdpp) after 6 hours. A whole-cell biocatalysis system with the strain (p01/AQ09) yielded 3.3 mM AQ after 18h (Fig. 2). These results demonstrated that inactivation of peptidases and the dipeptide transporter Dpp reduced the degradation of AQ, and thus increased AQ production.
Screening of BacD homologues
BacD is the key enzyme for AQ synthesis. We examined the known sequences annotated as L-amino acid α-ligase (BacD) in the NCBI database. According to the sequences of known or predicted BacD homologs, we selected a set of related sequences from different species, and constructed a phylogenetic tree of the previously reported BacD homologs and those used in this study (Fig. S1). The genes encoding BacD homologs from different species were codon-optimized by Nanjing Generay (China) and cloned into strain AQ10 (BW25113, ∆glnEB, ∆glsAB, ∆lpxM, ∆pepABDN, ∆dpp). The performance of different BacD proteins was investigated in vivo, using the respective strains as a whole-cell biocatalysts. The result showed that the strain overexpressing BaBacD (from Bacillus altitudinis) produced higher amount of AQ (19.2 mM) than strains with other BacD homologs. By comparison, 7.9 mM AQ was obtained using the strain overexpressing BsBacD (from Bacillus subtilis) (Fig. 3). Although there was soluble expression of BvBacD (from Beta vulgaris), VcBacD (from Vibrio campbellii), and SrBacD (from Streptomyces rubrolavendulae), only 3.0, 1.8, 0.5 mM AQ was respectively obtained(Fig. 3). BsaBacD (from Bacillus safensis), BloBacD (from Bifidobacterium longum subsp. infantis), PmBacD (from Perkinsus marinus), and PfBacD (from Pseudomonas fluorescens) were expressed as inclusion bodies (Fig. S2), and only a low amount of AQ was detected.
Construction of a glutamine synthesis module
To use the more readily available substrate glutamic acid, glutamine synthetase from Corynebacterium glutamicum (CgGlnA), which convert glutamic acid to glutamine was cloned into E. coli, resulting in the strain p00/BW25113. A final glutamine titer of 22.4 mM glutamine was obtained in the whole-cell bioconversion. During growth nitrogen replete growth conditions, glutamine synthetase adenylyltransferase/deadenylase (encoded by glnE) interacts with PII-1 protein encoded by glnB, which reduces the activity of glutamine synthetase, and glnE-glnB-deficiency was reported to lead to increased glutamine accumulation [23-25]. Therefore, a glnE-glnB-deficient strain expressing glnA (p00/AQ02) was constructed, and a glutamine titer of 27.8 mM was achieved, which was 24.1% higher than that of the strain p00/BW25113 (Fig. 4). In E. coli, glutamine was converts into glutamic acid by glutaminases GlsA (encoded by glsA) and GlsB (encoded by glsB), and further into α-ketoglutarate. Consequently, the glsA and glsB genes were deleted, resulting in strain AQ04 (ΔglsAΔglsB), which was then transformed with the plasmid p00, and the resulting strain p00/AQ04 produced 33.8 mM of glutamine after 18 hours of bioconversion. Further, glnE, glnB, glsA, glsB and lpxM were sequentially knocked out and 46.5 mM glutamine was accumulated by the resulting strain p00/AQ06, with a conversion rate of 93.0 % (Fig. 4). Consequently, the strain p00/AQ06 was used for further engineering.
Combination the AQ and glutamine synthesis modules
To achieve AQ production from glutamic acid and alanine, the AQ and glutamine synthesis modules were combined. The strain AQ10 was obtained by knocking out the genes pepN, pepA, pepB, pepD, dpp, glnE, glnB, glsA, glsB and lpxM. In AQ10, the degradation of AQ was alleviated, glutamine catabolism was effectively weakened as well. After introducing BsBacD, the resulting strain p01/AQ10 produced 7.9 mM AQ, which was four times more than the production of the original strain p01/BW25113 (Fig. 2).
The engineered strains with the plasmid p11 (pYB1s-CgglnA-BsbacD), co-expressing BsBacD and CgGlnA was used as a whole-cell biocatalyst for AQ production from alanine and glutamic acid. Removal of the peptidases PepA, PepB, PepD, and PepN, together with knocking out the transporter Dpp significantly increased AQ production, and a titer of 17.9 mM was produced by the strain AQ09 harboring p11 (Fig. 5). Due to the downregulation of glnE-glnB and glsAglsB, the biosynthesis of glutamine was enhanced, which resulted in increased AQ production, leading to a product titer of 29.8 mM in the strain AQ10 harboring p11 (Fig. 5). Inactivation of peptidases alleviated AQ degradation, and removing the transporter Dpp promoted the efflux of AQ. The results showed that combination of the strategies of peptidases inactivation, knocking out the transporter Dpp, and enhancing the glutamine supply by downregulation of glnE-glnB and glsA-glsB greatly enhanced AQ production.
Balance of the two synthesis modules by regulating protein expression
To balance flux in the two synthesis module for the purpose of increasing AQ production, the expression of BacD and GlnA proteins was studied. To co-express BaBacD or BsBacD with CgGlnA in different order, four plasmids p11 (pYB1s-CgglnA-BsbacD), p12 (pYB1s-BsbacD-CgglnA), p13 (pYB1s-CgglnA-BabacD), and p14 (pYB1s-BabacD-CgglnA) were constructed (Fig. 6c), and used to individually transform the host AQ10. Either L-amino acid α-ligase or glutamine synthetase was poorly expressed when BaBacD was co-expressed with CgGlnA (Fig. S3), leading to decreased AQ production. However, when CgGlnA was co-expressed with BsBacD, both proteins were expressed at high levels, and contributed to an increased yield of AQ after 18h of bioconversion. The AQ titer reached 29.8 mM when CgglnA was inserted in front of BsbacD (p11/AQ10), compared to 22.3 mM when CgglnA was expressed behind BsbacD (p12/AQ10) (Fig. 6a). The concentration of the intermediate metabolite, glutamine, in p11/AQ10 (22.8 mM) was higher than in p12/AQ10 (12.0 mM) (Fig. 6a). SDS-PAGE analysis of protein expression (Fig. S3) and the concentration of glutamine suggested that higher soluble expression of CgGlnA enhanced the supply of glutamine, and increasing the expression of BsBacD might further improve the synthesis of AQ.
In order to enhance the expression of BsBacD, its native RBS was replaced to upregulate the mRNA translation initiation rate in the recombinant strain. The translation rate prediction and design of new RBS was done using RBS Calculator 2.0[27-29]. The strain p15/AQ10 expressed more BsBacD protein (Fig. S4), and its AQ production increased by 76.1 % compared to p11/AQ10 (Fig. 6b).
Optimization of the conditions for whole-cell biocatalysis
After successfully constructing an engineered E.coli strains for AQ production by metabolic engineering, we investigated its applicability as whole-cell biocatalyst for the biotechnological production of AQ. Bioconversion parameters that affect the activity of the biocatalyst, such as temperature and pH, were investigated. AQ production reached maximal values at 30℃ (Fig. 7a) and pH 9.0 (Fig. 7b). A decreased in pH was observed as the bioconversion proceeded, which affected the biosynthesis of AQ. It should be noted that glucose was supplemented in the reaction mixture to supply ATP for the reactions catalyzed by GlnA and BacD, it was reported that excess glucose can lead to acetate accumulation. Consequently, we measured the concentration of acetate and found that it was accumulated. To alleviate this, different glucose feeding strategies were applied to reduce acetate accumulation in the bioconversion process, including 1) 50 mM glucose at once; 2) 10 mM every 3 hours; and 3) 20 mM every 3 hours. When a low concentration of glucose (10 mM) was fed every three hours (Fig. 7c), glucose was fully utilized (Fig. S5a), and only a small amount of acetic acid accumulated (Fig. S5b ), indicating that 10mM glucose fed every three hours matched AQ productivity. The time profiles of the bioconversion indicated that alanine was exhausted first, and the ratio of glutamic acid to alanine was investigated (Fig.7d). Under feeding with 10 mM glucose every three hours at 30 ℃ and pH 9.0, the strain p15/AQ10 produced 71.7 mM AQ, from 100 mM glutamic acid and 125 mM alanine, after 18 hours of reaction, corresponding to a productivity of 3.98 mM/h. Moreover, a conversion rate of 71.7% was achieved for glutamic acid representing a 100% increase compared to the conversion rate before the optimization.