Construction of the AQ Synthesis Module
E.coli strain p01/BW25113 expression BacD from Bacillus subtilis (BsBacD) which catalyzes the formation of AQ from alanine and glutamine was constructed for production of AQ, 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], inactivation of them might reduce AQ degradation. It has been reported that a transportation system coded by the dpp operon which is responsible for import of dipeptides, and deletion of dpp increase AQ accumulation [22]. By knocking out the genes pepN, pepA, pepB, pepD and dpp, the strain (AQ09), the degradation of AQ was alleviated. In starting host BW25113, 20 mM AQ was completely degraded at 3 hours, while only 1.3 mM in the chassis AQ09 after 6 hours. 3.3 mM AQ was obtained by the strain (p01/AQ09) in a whole-cell catalytic system after 18 h (Fig.2). The result demonstrate inactivation of peptidases and dipeptide transport system Dpp reduce the degradation of AQ and thus increase AQ production.
Screening of BacD with excellent properties
BacD is a key enzyme involved in AQ synthesis, BacD with efficient catalytic properties is screened. BacD from different species was codon optimized and synthesized by Nanjing Generay (China) and then cloned in strain AQ10 (BW25113, ∆glnEB, ∆glsAB, ∆lpxM, ∆pepABDN, ∆dpp) separately, including Bacillus altitudinis, Bacillus subtilis, Beta vulgaris, Bifidobacterium longum subsp. Infantis, Perkinsus marinus, Pseudomonas fluorescens, Bacillus safensis, Vibrio campbellii, Streptomyces rubrolavendulae. The strains was used in the whole-cell bioconversion for AQ synthesis. The result showed that strain with BaBacD (from Bacillus altitudinis) produced higher amount of AQ (19.2 mM) than strains with other BacDs. While 5.6 mM AQ was obtained by strain with BsBacD (from Bacillus subtilis) (Fig.3). Although, BvBacD (from Beta vulgaris), VcBacD (from Vibrio campbellii), SrBacD (from Streptomyces rubrolavendulae) were well-expressed but only 3.0 mM, 1.8 mM, 0.5 mM AQ was obtained separately (Fig.3). BsaBacD (from Bacillus safensis), BloBacD (from Bifidobacterium longum subsp. Infantis), PmBacD (from Perkinsus marinus), PfBacD (from Pseudomonas fluorescens) were poor expressed and very low amount of AQ was detected.
Construction of glutamine synthetic module
To use the more readily available substrate glutamic acid, glutamine synthetase from Corynebacterium glutamicum (CgGlnA) which convert glutamic acid to glutamine was cloned in E. coli resulted in the strain p00/BW25113. 22.4 mM glutamine was obtained in the whole-cell bioconversion by p00/BW25113. During nitrogen-rich growth, glutamine synthetase adenylyltransferase /deadenylase which encoded by glnE interacts with PII-1 protein encoded by glnB, jointly reduce the activity of glutamine synthetase, the glnE-glnB deficiency resulted in an increase glutamine accumulation [23-25]. The glnE-glnB-deficient strain (△glnE△glnB) expressing glnA was constructed (p00/AQ02), and 27.8 mM glutamine was achieved which was 24.1% higher than that of wild-type strain (Fig.4). In E. coli, glutamine degrades into glutamic acid by glutaminase GlsA (encoded by glsA) and GlsB (encoded by glsB)[26], and further decomposed into α-ketoglutarate. glsA and glsB were deleted in E.coli resulted in strain AQ04 (△glsA△glsB), which was then transformed with plasmid p00. 33.8 mM was obtained by strain p00/AQ04 exhibited increased glutamine production after 18 hours of bioconversion. Further, glnE, glnB, glsA, glsB and lpxM which involved in one of the last two acylation reactions needed to synthesize KDO2-lipid A in E.coli was knockout, and 46.5 mM glutamine was accumulated with a conversion rate of 93.0 % (Fig.4). p00/AQ06 was used in the thereafter study.
Combination of AQ synthetic module and glutamine synthetic module
To achieve AQ production from glutamic acid and alanine, AQ synthetic module and glutamine synthetic module was combined. The strain AQ10 was obtained constructed by knocking out the genes pepN, pepA, pepB, pepD , dpp, glnE, glnB, glsA, glsB and lpxM, the degradation of AQ was alleviated, glutamine catabolism was effectively weakened as well. And 7.9 mM AQ was obtained by the strain (p01/AQ10), which was four times as much as the original strain p01/BW25113 (Fig.2).
Plasmid co-expression BsBacD and CgGlnA was transformed in host strain AQ10. The engineered strain was used in the whole-cell biocatalysis for AQ production from alanine and cheaper substrate glutamic acid.
And 29.8 mM AQ was obtained (Fig.5) , which was much higher than that of p11/AQ09,p11/AQ06,p11/AQ04,p11/BW25113. The result showed that glnE-glnB deficiency and glutaminase glsA, glsB knock-out enhancing glutamine supply and the inactivation of peptidases PepA, PepB, PepD, PepN alleviate AQ degradation and transporter Dpp knock-out promote the efflux of AQ, all of that contributed to enhanced AQ production.
Regulation of the two module by balancing the protein expression
To regulation of the two module flux for improved AQ synthesis, the protein expression of BacD and GlnA was studied. BaBacD or BsBacD was co-expressed with CgGlnA. Four plasmids p11 (pYB1s-CgglnA-BsbacD), p12 (pYB1s-BsbacD-CgglnA), p13 (pYB1s-CgglnA-BabacD), p14 (pYB1s-BabacD-CgglnA) were constructed (Fig.6c), and transformed into the host AQ10 separately. Both of L-Amino Acid α-Ligase and glutamine synthetase were poorly expressed when BaBacD was co-expressed with CgGlnA, and led to a less AQ produced. However, when CgGlnA was co-expressed with BsBacD, both of the protein were well-expressed (Fig.s4), and contributed to higher yield of AQ after 18h of bioconversion, that is 29.8 mM when CgglnA was inserted in front of BsbacD (p11/AQ10) and 22.3 mM when CgglnA was expressed behind BsbacD (p12/AQ10), respectively (Fig.6a). 22.8 mM intermediate product glutamine was detected when CgglnA was put first, higher than that BsbacD was put first, which suggested that the well-expression of CgGlnA could ensure adequate supply of glutamine. The result hinted that increasing the expression of BsBacD might be tried to further improve the synthesis of AQ.
In order to enhance BsBacD expression, RBS of BsbacD was replaced to upregulate targeted mRNA's translation initiation rate in recombinant strain. Predicting translation rate and designing new RBS was carried out by RBS Calculator 2.0[27-29]. p15/AQ10 expressed more BsBacD (Fig.s5), AQ production increased 76.1% compared to p11/AQ10 (Fig.6b).
Optimization of the conditions of whole cell biocatalysis
The whole-cell biocatalytic conditions were investigated, including temperature, pH, glucose feeding and ratio of substrates. AQ production hit a high point at 30℃ (Fig.7a) and pH 9.0 (Fig.7b). For synthesis a molecules of AQ, two molecules of ATP are needed, because BacD and GlnA are ATP dependent enzymes. ATP can be supplied by oxidation of glucose. It was known that, if there is too much glucose, acetic acid is accumulated. So three strategies of batch flow glucose was tried, 1) 50 mM glucose at once; 2) 10 mM every 3 hours; 3) 20 mM every 3 hours. When low concentration of glucose (10 mM) was fed every three hours (Fig.7c), glucose was fully utilized (Fig.7d), only a small amount of acetic acid accumulated (Fig.7e ), which hint that 10mM glucose fed every three hours matched AQ productivity. 65.6 mM AQ obtained with productivity of 7.29 mM/h and conversion rate of 65.6%after 9 hours of reaction, which is twice as high as before, by p15/AQ10, from 100 mM glutamic acid and 125 mM alanine, fed 10 mM glucose every three hours, at 30 ℃ and pH9.0 (Fig.7f).