Escherichia coli BL21(DE3) optimized deletion mutant as the host for whole-cell biotransformation of N‑acetyl‑d‑neuraminic acid

N‑Acetyl‑d‑neuraminic acid (Neu5Ac) is the crucial compound for the chemical synthesis of antiflu medicine Zanamivir. Chemoenzymatic synthesis of Neu5Ac involves N-acetyl-d-glucosamine 2-epimerase (AGE)-catalyzed epimerization of N-acetyl-d-glucosamine (GlcNAc) to N-acetyl-d-mannosamine (ManNAc), and aldolase-catalyzed condensation between ManNAc and pyruvate. Host optimization plays an important role in the whole-cell biotransformation of value-added compounds. In this study, via single-plasmid biotransformation system, we showed that the AGE gene BT0453, cloned from human gut microorganism Bacteroides thetaiotaomicron VPI-5482, showed the highest biotransformation yield among the AGE genes tested; and there is no clear Neu5Ac yield difference between the BT0453 coupled with one aldolase coding nanA gene and two nanA genes. Next, Escherichia coli chromosomal genes involved in substrate degradation, product exportation and pH change were deleted via recombineering and CRISPR/Cas9. With the final E. coli BL21(DE3) ΔnanA Δnag ΔpoxB as host, a significant 16.5% yield improvement was obtained. Furthermore, precursor (pyruvate) feeding resulted in 3.2% yield improvement, reaching 66.8% molar biotransformation. The result highlights the importance of host optimization, and set the stage for further metabolic engineering of whole-cell biotransformation of Neu5Ac.

Introduction  is the main intermediate for the chemical synthesis of antiflu medicine Zanamivir (von Itzstein 2007).Classical Neu5Ac preparation methods include raw material extraction and chemical synthesis.However, due to the complicated extraction and downstream purification processes, excess use of large amount of organic solvents, and low purity of final product, the two methods are not suitable for large scale production.Chemoenzymatic synthesis, characterized by low solvent use, ambient reaction temperature, and high purity of product, provides a possible choice.
There are two pathways for the chemoenzymatic synthesis of Neu5Ac.The first is the two-step synthetase pathway which contains Abstract N-Acetyl-d-neuraminic acid (Neu5Ac) is the crucial compound for the chemical synthesis of antiflu medicine Zanamivir.Chemoenzymatic synthesis of Neu5Ac involves N-acetyl-d-glucosamine 2-epimerase (AGE)-catalyzed epimerization of N-acetyl-d-glucosamine (GlcNAc) to N-acetyld-mannosamine (ManNAc), and aldolase-catalyzed condensation between ManNAc and pyruvate.Host optimization plays an important role in the whole-cell biotransformation of value-added compounds.In this study, via single-plasmid biotransformation system, we showed that the AGE gene BT0453, cloned from human gut microorganism Bacteroides thetaiotaomicron VPI-5482, showed the highest biotransformation yield among the AGE genes tested; and there is no clear Neu5Ac yield difference between the BT0453 coupled with one aldolase coding nanA gene and two nanA genes.Next, Escherichia coli chromosomal genes involved in substrate degradation, product exportation and pH change were deleted via recombineering and CRISPR/Cas9.With the final E. coli Supplementary Information The online version contains supplementary material available at https:// doi.org/ 10. 1007/ s10529-023-03426-3.-acetyl-d-glucosamine (GlcNAc) to N-acetyl-d-mannosamine (ManNAc) and synthetase-catalyzed condensation between ManNAc and phosphoenolpyruvate (PEP) (Tabata et al. 2002;Yan and Fong 2018).The second is the two-step aldolase pathway (Fig. 1) which involves the same epimerization as that of the synthetase pathway, followed by an aldolase-catalyzed condensation between ManNAc and pyruvate (Kragl et al. 1991;Mahmoudian et al. 1997;Maru et al. 1998).
The synthetase pathway uses expensive PEP as substrate and yield is currently 15 g/l of Neu5Ac (0.14 g Neu5Ac/g GlcNAc) (Zhu et al. 2017); by contrast, the aldolase pathway uses inexpensive GlcNAc and pyruvate as substrates; and the yield is high up to 193.3 g/l of Neu5Ac (0.85 g Neu5Ac/g GlcNAc) for commercial production (Maru et al. 1998), thereby becoming the main research area.
Purified enzyme-mediated synthesis of Neu5Ac was reported years ago (Kragl et al. 1991;Maru et al. 1998), yet the expensive enzyme isolation and purification steps are not cost-effective.Whole-cell biotransformation, by using induced cell culture as catalyst, provides an inexpensive and simple alternative and has become a routine strategy for the production of value-added compounds (Wu et al. 2021).Wholecell biotransformation was also investigated for the production of Neu5Ac (Lee et al. 2007).
Escherichia coli aldolase gene nanA, due to its high expression and activity, is the main aldolase gene used.On the other hand, a variety of AGE genes were reported, including pRnBP (Maru et al. 1996), hRnBP (Takahashi et al. 1999), slr1975 (Tabata et al. 2002), bAGE (Lee et al. 2007), BoAGE2 (Sola-Carvajal et al. 2012), AvaAGE (Klermund et al. 2013), and all3695 (Lin et al. 2013).Recently we cloned and characterized a novel AGE gene BT0453 from human gut microorganism Bacteroides thetaiotaomicron VPI-5482 (ATCC 29148).BT0453 showed the highest protein solubility among the AGEs tested; biotransformation molar yield via coupled BT0453 and NanA reached around 50%, suggesting its potential in industrial production (Gao et al. 2019).The direct comparison of the biotransformation molar yield of the AGE genes has not been performed thus far.
Host genome engineering was reported to improve product yield.A GlcNAc producing strain was engineered by deleting man operon (manXYZ) and nag operon (nagCABE) to prevent GlcNAc degradation (Deng et al. 2005).Recently, the ldhA gene encoding lactate dehydrogenase and the poxB gene encoding pyruvate oxidase were deleted for pyruvate producing strain construction (Moxley and Eiteman 2021).
In this study, the biotransformation yields of three AGE genes, BT0453, slr1975 from Synechocystis sp.PCC6803 (Tabata et al. 2002), and bAGE from Anabaena sp.CH1 (Lee et al. 2007), were compared via single-plasmid whole-cell Neu5Ac biotransformation system.Then, an optimized host was obtained via E. coli BL21(DE3) chromosomal gene deletions.Precursor (pyruvate) feeding further improved the yield.The research highlights the importance of optimizing biotransformation host for Neu5Ac production.
endonuclease I-SceI to cure of the plasmids after genome editing.

Vector construction
Molecular biology manipulations were performed as per the standard protocols (Sambrook and Russell 2001).Vector construction steps are provided in Supplementary information.The PCR primers and gene deletion oligonucleotides are listed in Supplementary Table S2.

Chromosomal gene deletions
Deletions of the nan operon (nanKETAR ), man operon, nag operon, and poxB gene were performed via coupling of recombineering and meganuclease I-SceI (Kuhlman and Cox 2010;Posfai et al. 2006).Briefly, for deletion the gene of interest (GOI), targeting DNA consisting of 50 bp homologous arm upstream and downstream (A and C), two I-SceI cleavage sites, kanamycin resistance gene (neo) and 40 bp B sequence behind C was amplified.The targeting DNA was electroplated into annealaseproficient electrocompetent cells (Murphy 2016), GOI was replaced by the targeting cassette under kanamycin selection via recombineering.After sequence confirmation, I-SceI was induced and cut at its cleavage sites, creating double strand break which was repaired by strain's inherent recombination-mediated recombination between the two 40 bp B sequences, generating the deletion mutant.Schematic presentation of the recombineering and I-SceI-mediated gene deletion is shown in Supplementary Fig. S1.The genotypes and sequencing results of the nan, man, nag, and poxB gene deletion mutants are shown in Supplementary Figs.S2,   S3, S4 and S5, respectively.The ldhA and ackA gene deletions were performed via coupling of ssDNA recombinering and CRISPR/Cas9 (Jiang et al. 2013;Oh and van Pijkeren 2014).In brief, coelectroporation of sgRNA expression plasmid and deletion oligo into the annealase and Cas9-proficient electrocompetent cells harboring pLS3535 (Li et al. 2019) resulted in the CRISPR/Cas9-assisted, recombineering-mediated gene deletion.The edited clone was subsequently transformed with a p15A backbone-based, Km R plasmid pLS3550 (Li et al. 2019)  Whole-cell biotransformation, precursor feeding, and Neu5Ac crystallization Neu5Ac whole-cell biotransformation was carried out as reported (Gao et al. 2019) by using 20 ml (OD 600 ~ 20.0) of the E. coli cells harboring singleplasmid cloned with AGE and nanA gene or each 10 ml (OD 600 ~ 20.0) of the E. coli cells harboring plasmid clone with AGE or nanA gene (two-plasmid system), respectively.Each 20 μl (OD 600 ~ 12.0) aliquot was sampled at 12 h, 24h, 36h, and 48 h and subjected to HPLC for Neu5Ac assay as described (Mahmoudian et al. 1997).Two kinds of pyruvate feeding were conducted.For one time feeding, the initial reaction consisted of 0.8 M GlcNAc and 1.2 M pyruvate; after 24 h, pyruvate was fed to a final concentration of 1.67 M. For twice feeding, the initial reaction consisted of 0.8 M GlcNAc and 0.5 M pyruvate with pyruvate was fed at the 24 h and 36 h, reaching a final concentration of 1.2 M and 1.67 M, respectively.Neu5Ac crystallization was performed as reported (Maru et al.1998).A small portion of the refined Neu5Ac powder was dissolved in ddH 2 O and analyzed by HPLC to determine the Neu5Ac purity and refinement yield.

Comparison of AGE genes for the biotransformation molar yield of Neu5Ac
During the characterization of BT0453, it was found that the yield of two-plasmid system showed around 10% higher than that of the single-plasmid system (Gao et al. 2019).However, as single-plasmid system is much more convenient to manipulate, thus it was selected for the comparison among AGE genes.Three AGE genes (BT0453, slr1975, and bAGE) were selected, in which BT0453 is the most soluble protein, while slr1975 and bAGE represent the two most often used AGEs in the whole-cell biotransformation of Neu5Ac.Firstly, for cloning convenience, restriction enzyme recognition cloning sites were added to single BT0453-nanA expression vector pLS2845 (Gao et al. 2019), obtaining pLS4106.Then, based on pLS4106, a series of single-plasmid expression vectors was constructed by coupling AGE gene with one nanA or two copies of nanA genes, obtaining BT0453-2 nanA (pLS4109), slr1975-nanA (pLS4111), slr1975-2 nanA (pLS4112), bAGE-nanA (pLS4114), and bAGE-2 nanA (pLS4115).See Supplementary Table S1 for detail.
Each plasmid was transformed into E. coli LS2802 (E. coli BL21(DE3) ΔnanA) (Gao et al. 2019) and whole cells with induced expression of AGE and NanA were used for Neu5Ac biotransformation.As seen in Fig. 2, E. coli LS2802/pLS2845 and E. coli LS2802/pLS4106 showed nearly the same GlcNAc molar transformation yield (~ 47.3%); and BT0453 was the best AGE gene among the genes tested.Note that at 12 h and 24 h time points, the BT0453-2 nanA (pLS4109) yield was higher than that of BT0453-nanA (pLS4106), while at 36 h (the time point with highest yield of Neu5Ac), no difference was shown for BT0453-nanA and BT0453-2 nanA, implying that though BT0453-2 nanA biotransformation velocity was much faster, and the enzyme activities reached the plateau at 36 h.For slr1975 and bGAE, yield improvement was observed for two nanA genes compared with one nanA gene (7.5% and 5.6% improvement, respectively); however, unlike the results previously reported (Kao et al. 2018), no drastic improvement was observed.

E. coli host strain optimization
After AGE gene screening, a variety of chromosomal genes were deleted to optimize the E. coli biotransformation host.The E. coli LS2802 was used for characterization of BT0453 and biotransformation yield comparison among AGE genes.As the first step to optimize the host, we compared the wild-type E. coli BL21(DE3) strain and E. coli LS2802.No clear yield difference was observed between them (shown as WT and ΔnanA in Fig. 3, respectively); however, to eliminate the background NanA activity which may hinder further comparison, E. coli LS2802 was selected as the starter strain for following gene deletions.
Besides deletions of the man and nag operons to prevent GlcNAc degradation (Deng et al. 2005), and deletions of ldhA and poxB genes to prevent pyruvate degradation (Moxley and Eiteman 2021), the whole nan operon and the ackA gene were also deleted.The nan operon is involved in Neu5Ac degradation and exportation.The ackA gene encodes acetate kinase which takes part in the synthesis of acetic acid; as the optimized pH of Neu5Ac chemoenzymatic reaction including AGE and aldolase is around pH 8.0, deletion of ackA might avoid the generation of acetic acid leading to pH decrease, thus helpful for Neu5Ac production.(Jiang et al. 2013;Oh and van Pijkeren 2014).Initially the established combination of ssDNA recombineering and CRISPR/Cas9 method for E. coli chromosomal gene point mutation strategy was adapted to delete all the selected genes (Li et al. 2019); however, except ldhA and ackA, other four gene deletions were failed, which might be due to their large sizes (all four genes are larger than 1.2 kb), implying that 1.2 kb might be the upper size limit for highly efficient ssDNA recombineering and CRISPR/ Cas9-mediated gene deletion.Combined action of recombineering (Datsenko and Wanner 2000;Murphy 2016) and homing nuclease I-SceI was then successfully employed for the deletion of nan operon, man operon, nag operon, and poxB gene.Verifications of the genotypes of six gene deletion mutants through ethidium bromide stained agarose gel electrophoresis are shown in Fig. 4.
Each deletion mutant was transformed with pLS4106 and the resultant strains were cultured, induced with IPTG and used for whole-cell biotransformation of Neu5Ac.As seen in Fig. 3, deletion of Δnan exhibited no molecular transformation yield difference with ΔnanA; for Δman, ΔldhA, and ΔackA strains, even a slightly yield decrease was observed.The results indicated that nan operon-mediated Neu5Ac exportation, man operon-mediated GlcNAc exportation, ldhA-mediated pyruvate degradation, and AckA-mediated pH pertubation, played negligible influence on whole-cell Neu5Ac biotansformation.In contrast, ~ 9% and ~ 6% yield improvement were observed for Neu5Ac biotransformation with Δnag and ΔpoxB hosts, respectively, indicating that Δnag reduced GlcNAc degradation and ΔpoxB reduced pyruvate degradation.Combined Δnag and ΔpoxB was subsquently performed and the resultant strain E. coli LS4421 showed a synergistic improvement over Δnag and ΔpoxB single mutants (~ 8% and 12% yield improvement over Δnag strain and ΔpoxB strains, respectively), with yield being 63.6% (Fig. 3).
Whole-cell biotransformation with two-plasmid system biotransformation was followed by using plasmids pLS2824 and pLS2845 (Gao et al. 2019) with E. coli LS4421 as host.Unexpectedly, unlike E. coli LS2802 with which two-plasmid system showed 10% higher yield than single-plasmid system, with optimized E. coli LS4421 host strain, two-plasmid systems showed nearly the same yield as that of the single-plasmid pLS4106 system (63.6% vs. 64.2%).

Precursor feeding further improved biotransformation yield
Precursor feeding is a normally used fermentation strategy to improve the product yield.As described in "Materials and methods" section, one time and twice pyruvate feeding with single-plasmid and two-plasmid systems, and each with 1 ml and 10 ml reaction volumes, were tested with the final the pyruvate concentration reached 1.67 M. For both plasmid systems and for both the 1 ml and 10 ml reaction volumes, only slightly yield improvement (~ 1%) was observed via one time feeding; by contrast, around 3.2% yield improvement was observed for twice feeding.A final 66.8% GlcNAc molar transformation yield was obtained and the productivity was 4.58 g/l/h.HAc extraction and crystallization process were applied for the purification of Neu5Ac from the 10 ml reaction.After purification, 1.77 g GlcNAc resulted in 0.94 g Neu5Ac (around 99.0% purity).The HPLC analysis Fig. 3 E. coli BL21(DE3) serial gene deletion mutants harboring pLS4106 for whole-cell biotransformation of Neu5Ac.The gene deletion genotype was noted with E. coli BL21(DE3) was indicated as WT.nan: nan operon; man: man operon; nag: nag operon.The 50% molar transformation yield would get 0.12 g of Neu5Ac in 1 ml of reaction.Data are the averages from three independent experiments and error bars denote standard deviation of the biotransformation products and the purified products is shown in Supplementary Fig. S9.

Discussion
Due to its high demand and high price, inexpensive and high yield production route of Neu5Ac is urgently needed.Whole-cell biotransformation, by using induced cell culture as catalyst, provides an effective route and has become a routine strategy for the production of value-added compounds.Three AGE encoding genes (BT0453, slr1975 and bAGE) were compared for whole-cell Neu5Ac biotransformation.BT0453 shows 56% and 57% pair-wise similarity with slr1975 and bAGE, respectively; while slr1975 and bAGE shares 72% pair-wise similarity.Most of the reported AGEs show high similarity to the AGEs tested herein.For example, BoAGE2 shows 91% similarity with BT0453; AvaAGE and All3695 show 98% and 97% similarity with bAGE, respectively.The pRnBP (porcine renin-binding protein) and hRnBP (human renin-binding protein) share 87% similarity, the two genes were not selected as hRnBP showed < 10% overexpressed protein solubility (Gao et al. 2019), restricting further protein manipulation.BT0453 showed the highest transformation yield, corresponding to its high protein solubility.
The host's chromosome may contain genes which encode the enzymes that involved in the degradation of substrate or product, biosynthesis of small molecular which causes pH change, and biosynthesis of toxic products which hinders strain's survival by damaging stain's cell membrane, etc.Therefore, host strain optimization plays an important role in the biotransformation of final product.In this study, Fig. 4 Representative ethidium bromide stained agarose gels of the six gene deletion mutants.Colony PCR was performed from the genomic DNA of wild-type and the deletion mutant; and the resulting amplicons were subjected to DNA gel electrophoresis.A Genotyping of Δnan mutant.Lanes 1, 2, and 3 denote the s204F-s204R primers amplified PCR products from E. coli LS2802, E. coli LS4401 (Km R ), and E. coli LS4403 (Km S , Δnan), respectively.Lane 4 and 5 denote the s205F-s205R primers amplified nanT region PCR products from E. coli LS2802 and E. coli LS4403 (Km S , Δnan), respectively.No nanT region was left in the deletion mutant E. coli LS4403.M1, Trans2K Plus DNA molecular marker (Tiangen, China) with sizes are 8.0, 5.0, 3.0, 2.0, 1.0, 0.75, 0.5, 0.25, and 0.1 kb.B Genotyping of Δman mutant.Lanes 1, 2, and 3 denote the s205F-s205R primers amplified PCR products from E. coli LS2802, E. coli LS4404 (Km R ), and E. coli LS4406 (Km S , Δman), respectively.M2, DL5000 DNA molecular marker (Takara) with sizes are 5.0, 3.0, 2.0, 1.5, 1.0, 0.75, 0.5, 0.25, and 0.1 kb.C Genotyping of Δnag mutant.Lanes 1, 2, and 3 denote the s209F-s209R primers amplified PCR products from E. coli LS2802, E. coli LS4407 (Km R ), and E. coli LS4409 (Km S , Δnag), respectively.Lane 4 and 5 denote the s210F-s210R primers amplified nagB region PCR products from E. coli LS2802 and E. coli LS4409 (Km S , Δnag), respectively.No nagB region was left in the deletion mutant E. coli LS4409.M1, Trans2K Plus DNA molecular marker.D Genotyping of ΔpoxB mutant.Lanes 1, 2, and 3 denote the s214F-s214R primers amplified PCR products from E. coli LS2802, E. coli LS4413, and E. coli LS4415 (Km S , ΔpoxB), respectively.M3.DL2000 DNA molecular marker (Takara) with sizes are 2.0, 1.5, 1.0, 0.75, 0.5, 0.25, 0.1 kb.E Genotyping of ΔldhA mutant.Lanes 1, 2, and 3 denote the s211F-s211R primers amplified PCR products from E. coli LS2802, E. coli LS4412 (ΔldhA), respectively.M3.DL2000 DNA molecular marker (Takara).F Genotyping of ΔackA mutant.Lanes 1, 2, and 3 denote the s215F-s215R primers amplified PCR products from E. coli LS2802, E. coli LS4418 (ΔackA), respectively.M3.DL2000 DNA molecular marker (Takara).Arrows point the amplified fragments which were further validated via DNA sequencing the yield of whole-cell biotransformation yield of Neu5Ac was markedly improved via host strain optimization.Combined deletion of nag operon which is beneficial for GlcNAc biotransformation (Deng et al. 2005) and deletion of poxB which is beneficial for pyruvate biotransformation (Moxley and Eiteman 2021) was found to show the synergistic improvement effect for Neu5Ac production (up to 16.5%).The results presented herein suggest that host plays crucial roles in the biotransformation of value-added products, especially for those products which are negatively influenced by the enzymes encoding genes exist in the host's chromosome.All the obtained deletion mutants showed nearly the same growth characteristics (colony growth and LB liquid shake flask culture) as that of the wild-type (data not shown).Noted, however, the 4.58 g/l/h productivity, which was higher than the volumetric productivity of 0.79 g/l/h of pure enzyme biocatalysis (Maru et al. 1998), is still much lower than the productivity of 15.9 g/l/h reported (Kao et al. 2018).It is also worth noting that transformation yield decreased after 36 h, suggesting that the reaction equilibrium was achieved (both AGE-mediated epimerization and aldolase-mediated condensation are reversible reactions).
Highly efficient recombineering and CRISPR/ Cas9 techniques were used in the gene deletions, paving the way for optimization of host strain rapidly and conveniently.The single-plasmid biotransformation system was implemented for the Neu5Ac biotransformation in this research.The system might also be employed as a simple platform for the characterization of AGE genes to be explored in current bacterial genome or in metagenomic DNA.Directed evolution has been employed for the engineering of glucosamine synthase for the production of GlcNAc (Deng et al. 2006), as well as engineering of NanA for the production of Neu5Ac derivatives (Campeotto et al. 2010).Development of high throughput directed evolution strategies for AGE and NanA in whole-cell biotransformation of Neu5Ac would further explore the potential of BT0453, other AGEs, and NanA.Equally important is the fermentation studies including medium optimization, fermentation scale-up, and product extraction.
In summary, the study sets the stage for Neu5Ac process development via further exploration of AGE and nanA genes, over expression of key genes, direction evolution, and finally industrial production of Neu5Ac.

Fig. 1
Fig. 1 Schematic outline of the two-step biotransformation of Neu5Ac.AGE catalyzes the epimerization of inexpensive substrate GlcNAc to ManNAc, aldolase (NanA) catalyzes the condensation between ManNAc and pyruvate resulting in Neu5Ac resulting in the replacement of the λ-Red annealase and Cas9 expression plasmid pLS3535 under kanamycin selection.The sgRNA expression plasmid and pLS3550 (both harbor I-SceI inducible expression cassette and I-SceI cleavage sites) were cured via I-SceI induction and selection of Ap S Km S clones.Outline of the CRISPR/Cas9-assisted ssDNA recombinering-mediated gene deletion strategy is shown in Supplementary Fig. S6.The genotypes and sequencing results of the ldhA and ackA gene deletion mutants are shown in Supplementary Figs.S7 and S8, respectively.

Fig. 2
Fig. 2 Comparison of the yield of the whole-cell biotransformation of Neu5Ac among three AGE genes coupled with one nanA or two nanA genes.The 50% molar transformation yield would get 0.12 g of Neu5Ac in 1 ml of reaction.Data are shown as the averages from three independent experiments and error bars denote standard deviation