3.1 Modification of the key genes for L-homoserine accumulation
Several system metabolic engineering strategies have been explored for the biosynthesis of AFAAs in E. coli, such as improving the supply of precursors and cofactors and facilitating the efflux efficiency.[2, 18] Improving the supply of precursors is regarded as an important approach for the biosynthesis of L-homoserine, i.e. oxaloacetate (OAA), L-aspartate (L-ASP), Aspartyl-β-phosphate (Asp-P), and aspartyl-β-semialdehyde (Asp-SA). The OAA supply was considered as the bottleneck for the synthesis of AFAAs, and thus it was speculated that enhancing the OAA pool could facilitate L-homoserine production. Another strategy for increasing L-homoserine production is to overexpress the key enzymes to enhance the metabolic flux from OAA precursor toward the product of L-homoserine, especially these three enzymes: aspartokinase (AKI encoded by thrA, AKII encoded by metL, and AKIII encoded by lysC), aspartyl semialdehyde dehydrogenase (encoded by asd), and L-homoserine dehydrogenase (encoded by metL and thrA), as shown in Fig. 1. The ThrA and MetL are bifunctional enzymes with both aspartokinase and L-homoserine dehydrogenase activity [1, 19], which play essential roles in the synthesis pathway. The generation of NADPH as reducing equivalent is considered as a limiting step for optimizing the production of AFAAs. It can be found that two NADPH-dependent enzymatic reactions are involved in L-homoserine biosynthesis and two molecules of NADPH are needed to generate one molecule of L-homoserine (Fig. 1). Therefore, NADPH supply is a critical factor for L-homoserine production, which could be promoted by overexpressing the pntAB (encoding NAD(P) transhydrogenase) as described previously.[3, 18] Moreover, engineering the export system could facilitate the efflux of L-homoserine across the cell envelope to increase the extracellular product accumulation and alleviate the cellular metabolic burden.
Usually, L-homoserine cannot be accumulated in the wild type E. coli W3110 cells. In our previous work, LJL3 (ΔlysAΔmetAΔthrBC) was constructed by blocking the degradative and competing pathways to accumulate 0.2 g/L L-homoserine, which was chosen as the starting strain in the present work. The L-homoserine production was still very low due to the limited availability of precursors. In E. coli, the anaplerotic pathway catalyzed by phosphoenolpyruvate carboxylase (encoded by ppc) is considered to be an important source of OAA.[3, 24] Therefore, the plasmid pKK-ppc was constructed to overexpress the ppc gene, which was then transferred into LJL3, obtaining strain SHL1. The L-homoserine titer of strain SHL1 reached 1.0 g/L in shake flask medium at 44 h, which was 5.0-fold higher than that of strain LJL3. It was indicated that ppc overexpression resulted in an increased carbon flux from glucose to OAA and enhanced the OAA supply for L-homoserine production. In addition, the thrA, metL, and asd genes were introduced into pKK-miniPtac, resulting in plasmids of pKK-thrA, pKK-metL, and pKK-asd, and were then transferred into LJL3 to obtain strains of SHL2, SHL3, and SHL4, respectively. As shown in Fig. 2A, the strains SHL2, SHL3, and SHL4 produced 0.8 g/L, 3.3 g/L and 0.6 g/L of L-homoserine in flask cultures, respectively. The results indicated that overexpressing metL could significantly improve the accumulation of L-homoserine. Another target gene pntAB (encoding a NAD(P) transhydrogenase) was overexpressed using plasmid pKK-pntAB to improve the NADPH generation, obtaining strain SHL5. SHL5 harboring pKK-pntAB produced 1.2 g/L L-homoserine, which showed a good capacity of the NADPH supply for L-homoserine biosynthesis.
Previous reports showed that high level of L-homoserine could cause the toxicity stress, which inhibited the cell growth, the activity of NADP+-glutamate dehydrogenase, and L-homoserine production. [25, 26] Notably, the biomass of strains SHL2, SHL3 and SHL4 was significantly decreased due to the toxicity caused by increased accumulation of L-homoserine (OD600 from 9.9 to 7.5~8.3) (Fig. 2B). Efficient efflux of L-homoserine is an effective strategy to decrease the intracellular L-homoserine level, that can relieve the inhibition on cell growth. RhtA (encoded by rhtA) has been identified as the potential exporter of L-homoserine. To boost the efflux of L-homoserine, rhtA was overexpressed to obtain the strain SHL6 harboring pKK-rhtA, achieving a L-homoserine titer of 1.6 g/L, as shown in Fig. 2A. It was beneficial for relieving the toxicity stress arising from the L-homoserine accumulation.
Essentially, the increasement of metabolic flux causes a higher demand for precursor substrates. Our results indicated that strengthening the intracellular key enzyme expression level was necessary for enhanced production of L-homoserine. Liu et al. have strengthened thrA and metL expression level, and Mu et al. have overexpressed thrA and asd to improve the production of L-homoserine.[2, 18] Meanwhile, Mu et al. have revealed the redox balance is important for increasing the NADPH supply and is beneficial for improving bioproducts, such as fatty acids and succinate.[18, 27] This strategy can also be applied to L-homoserine production as regeneration of NADPH from NADH by overexpressing pntAB gene. In short, overexpression of genes in enhancing the precursor flux (i.e. the OAA and L-ASP supply), the NADPH generation and the efflux of L-homoserine was essential for L-homoserine production.
3.2. Strengthening the genes involved in L-homoserine biosynthesis at the genome
To improve the stability of engineered strains, overexpressing the key genes by moderating the E. coli LJL3 genome is feasible. Nowadays, CRISPR-mediated gene editing technology is beneficial for genome modification of microorganisms, owing to the high efficiency and seamless gene editing characteristics. To further improve the production of L-homoserine and constructing a stable cell factory, five genes (ppc, thrA, metL, and asd, and rhtA) were individually up-regulated by replacing the native promoter with a strong trc promoter in LJL3 genome using the CRISPR-Cas9 system. First, the ppc gene in LJL3 was chromosomally enhanced by replacing its native promoter with the trc promoter, resulting in SHL7 to increase the carbon flux into L-homoserine accumulation. After 48 h of fermentation, L-homoserine production by the SHL7 strain was increased by 75% compared with that of LJL3, reaching 0.35 g/L (Fig. 2C). Moreover, to convert L-aspartate to L-homoserine, the thrA gene was subsequently upregulated by replacing the native promoter with the trc promoter in the genome of SHL7, obtaining strain SHL8. The L-homoserine titer of SHL8 was 0.7 g/L, which was 2.0-fold higher than that of SHL7. Next, the native promoter of asd gene was in situ replaced with Ptrc to improve L-homoserine production, generating SHL9. As a result, the production of L-homoserine from flask cultivation of SHL9 strain was further increased to 0.9 g/L. To increase the NADPH pool, the promoter of pntAB driven by was also replaced with Ptrc, generating strain SHL10. After 48 h of cultivation, strain SHL10 achieved 1.2 g/L L-homoserine, which was 33.3% higher than that of SHL10 (Fig. 2C). To boost the efflux of L-homoserine, rhtA was overexpressed by replacing native rhtA promoter with Ptrc promoter of SHL10 genome, obtaining strain SHL11. L-homoserine production was significantly increased to 2.1 g/L in SHL11, 1.8-fold higher than that of SHL10 (Fig. 2C), which might be due to the higher transcription level of rhtA driven by the strong trc promoter. Moreover, the biomass (OD600) was also improved from 10.4 to 12.3, 18.3% higher than that of SHL10, which was consistent with the production performance (Fig. 2D). The results showed that enhancing the efflux of L-homoserine facilitated both L-homoserine production and cell growth. Herein, the CRISPR-Cas9 mediated in situ promoter replacement was successfully applied in enhancing the expression of ppc, thrA, asd, pntAB and rhtA at the genome level, which was beneficial for the L-homoserine production. Nevertheless, the overall production of L-homoserine in the engineered strain was still low, and the increment was not significant as the overexpression at the plasmid level.
Considering the essential role of the bifunctional enzyme of MetL, the corresponding metL gene was overexpressed in SHL11 with the plasmid pKK-metL, generating the strain SHL12. As a result, the titer of 6.2 g/L was achieved for L-homoserine in SHL12, which was 3.0-fold higher than that of SHL11. It was proved that metL was the crucial gene to channel more carbon flux from glucose to L-homoserine, and the increased ASP-P pool was beneficial for improving the production of L-homoserine. However, enhancing the plasmid stability of pKK-metL was still an important issue due to the potential toxicity of L-homoserine. SHL12 cells begin to lose the plasmids after several rounds of division without any selection pressure (Fig. S1). When antibiotics are added, cells could harbor plasmids to obtain the resistance to the antibiotic. The plasmids can be unevenly distributed due to degradation or the shortage of selection pressure. Thus, exploring strategies to maintain stability of pKK-metL plasmid without antibiotics addition was important for L-homoserine production.
3.3 Constructing auxotrophic complementation and hok/sok systems to improve the plasmid stability
Losing plasmid represents a major problem in large-scale cultivation of bacteria carrying plasmids, which are commonly used for overexpression of cloned genes. Several approaches have been conducted to enhance plasmid stability.[9, 11, 28] Herein, auxotrophic complementation and hok/sok systems were constructed to maintain the stability of pKK-metL plasmid. The auxotroph biosynthetic system, combined with the complementation of essential genes, could enable the stable maintenance of plasmid without the addition of antibiotics, because the essential genes should be expressed by plasmid instead of chromosome to ensure the cell growth. Moreover, choosing the proper gene directly related to cell viability could tightly control the plasmid copy number depending on its expression level and thus minimize cell-to-cell variations during antibiotic-free cultivation.[9, 29] L-aspartate, the precursor of L-homoserine, is one of the most important 4-carbon amino acid platform compounds.[30, 31] As shown in Fig. 3A, the L-aspartate-amino-acid synthesis genes aspA and aspC encode aspartate aminotransferase, which could transfer fumarate and oxaloacetate to the key precursor of L-aspartate for L-homoserine, respectively. An auxotrophic complementation system was developed by deleting the chromosomal copies of these aspartate-amino-acid synthesis genes in the SHL11 genome to obtain SHL14 (Fig. 3B). The aspA and aspC genes were subsequently introduced into pKK-metL plasmid using a strong trc promoter and the native promoter to construct pKK-metL-aspA-aspC-1 and pKK-metL-aspA-aspC-2, respectively (Fig. 3B). The engineered strain SHL16 and SHL17 containing pKK-metL-aspA-aspC-1 and pKK-metL-aspA-aspC-2 were constructed, compared with the control strain SHL15 containing pKK-metL (Fig. 3B), respectively. The daughter cells were considered to be forced to maintain the plasmids as there was no L-ASP feeding in fermentation medium, making the cells totally dependent on the maintenance of plasmid. Unexpectedly, the titer of SHL16 was dramatically decreased to 2.2 g/L, while the titer of SHL17 was slightly higher than SHL16 (2.5 g/L), both lower than that of the control stain SHL15 (Fig. 3C). Thus, constructing the auxotrophic complementation system to maintain plasmid stability was failed herein. It was speculated that the overexpression of aspA amd aspC genes might affect the expression resources for metL gene, thus decreased the expression level of MetL and the L-homoserine production.
Plasmid instability is mainly caused by the factors leading to an uneven distribution of plasmids to the daughter cells during cell division. To improve the plasmid stability, the hok/sok system, one of the toxin/antitoxin systems, was employed to maintain the stability of plasmid pKK-metL herein. It was assumed that the plasmid-lacking daughter cells could be killed via a system of stable toxins and unstable antitoxins, both expressed from the plasmid (Fig. 4A). The hok/sok gene cassette was cloned into two different locus of pKK-metL to investigate the effect of locations on gene expression and plasmid stability (Fig. 4B). One locus was next to the gene metL (generating plasmid pKK-metL-hok1), and the other locus was beside the antibiotic gene amp (generating plasmid pKK-metL-hok2). Then, the control strain of SHL12 (containing pKK-metL), two hok/sok constructed strains SHL18 (containing pKK-metL-hok1) and SHL19 (containing pKK-metL-hok2) were cultured in shake flasks without Amp (50 mg/L), respectively. Among these three strains, only SHL19 could retain the L-homoserine production regardless of the antibiotics added or not. As shown in Fig. 5A, the plasmid losing rate of strain SHL12 cultured without antibiotics was 94.0%. The plasmid losing rate of SHL19 (pKK-metL-hok2) cultured without antibiotics was 5.2%, which was much lower than that of SHL12 (Fig. 5A). The L-homoserine production of SHL19 was 6.1 g/L, which was similar with strain SHL12 (6.2 g/L). Single colony of SHL19 was inoculated into 3 bottles flask fermentation medium in parallel by 10 fermentation batches, respectively. As a result, all fermentation batches of SHL19 (10/10) could maintain the titer of 6 g/L (Fig. 5B).
The hok/sok system is a type I toxin/antitoxin system, which could enhance plasmid stability by the antisense RNA post-segregationally killing. The half-life of hok mRNA is longer than that of the sok RNA. Thus, the daughter cells losing the plasmid would die during division since the hok mRNA translated to the toxin protein (Fig. 4A).  On the other hand, the daughter cells with plasmid will survive if the sok antisense RNA binding with hok mRNA to generate a complex that can be decayed by RNase III (Fig. 4A). [32-34] Introducing the hok/sok system into plasmid pKK-metL might make the cell growth dependent on the maintenance of plasmid, resulting in the improved plasmid stability without the selection pressure of antibiotics. The results indicated that the hok/sok system was beneficial for maintaining plasmids for L-homoserine production in E. coli.
3.4. Fed-batch fermentation for L-homoserine in a 5 L fermenter
To evaluate the productivity of strain SHL12 (harboring pKK-metL) and SHL19 (harboring pKK-metL-hok2) for L-homoserine production, the fed-batch fermentation was implemented in a 5 L fermenter with 2.5 L working volume without antibiotics addition. As shown in Fig. 6, the cell growth steadily increased, and the maximum OD600 of strain SHL19 reached 68 at 48 h, while the OD600 of SHL12 merely reached 48. The growth of strains SHL12 and SHL19 showed different trend: the OD600 of SHL12 reached the maximum at 23.5 h and decreased at 35 h, while the OD600 of SHL19 continuously increased. The accumulation of L-homoserine in strain SHL19 was initially detected at 4 h, and finally reached the maximum titer of 44.4 g/L at 48 h, which was 19.7% higher than that of SHL12 (35.1 g/L). It was indicated that the introduction of hok/sok system into strain SHL19 was beneficial for the maintenance of plasmid and L-homoserine production during the fed-batch fermentation. The productivity of L-homoserine in strain SHL19 was 0.93 g/L/h, which was 1.26-fold higher than that of SHL12 (0.73 g/L/h). Compared with the L-homoserine producing strain LJL (0.82 g/L/h) reported previously, SHL19 showed higher productivity in fed-batch fermentation. The titer and productivity of L-homoserine obtained herein were lower than the two works reported in this year.[3, 18]
Nevertheless, there was no antibiotics added in this work, which could be beneficial for the environment. The production of L-homoserine could be further improved combing with other strategies, such as a dynamical controlling degradation system and a balancing redox strategy. Moreover, low OD600 value (up to 68) was obtained in the fed-batch fermentation. Additional modifications of fermentation feeding strategy was also needed to optimize fermentation condition for higher production of L-homoserine.
3.5. Ectoine biosynthesis facilitated with the hok/sok system
Ectoine is widely used in skin protection, pharmaceutics and cosmetics, which is an important derivative from AFAAs synthesis pathway using the same precursor ASP-SA with L-homoserine. Ning et al. constructed the engineered E. coli strain with an ectoine titer of 25.1 g/L by introducing the ectABC gene cluster from Ectothiorhodospira halochloris, weakening the competitive pathway, releasing feedback resistant and improving the oxaloacetate pool. To test the application of hok/sok system for AFAAs synthesis, this system was applied for the ectoine production herein. First, the ectoine biosynthesis strain was constructed by introducing the ectABC cluster into E. coli MG1655, generating strain S00. The resultant S00 strain synthesized 1.8 g/L ectoine in the flask fermentation. Second, several metabolic engineering strategies were used by modifying the E. coli MG1655 genome to obtain metabolic engineering strain: improving the precursor OAA level by replacing the phosphoenolpyruvate carboxylase (ppc) promoter with a trc promoter to getting strain S01; enhancing the oxaloacetate supply by deleting the transcriptional repressor (encoded by iclR) to promote glyoxylate shunt to obtain strain S02; and concentrating the carbon flux from L-aspartate-β-semialdehyde to ectoine by deleting the bifunctional aspartokinase/homoserine dehydrogenase (encoding by thrA) to generating strain S03 (Fig. 7A). As shown in Fig. 7B, the ectoine titer of S03 containing plasmid ptrc99a-ectABC was increased to 3.0 g/L, which was 1.7-fold higher than that of the parent strain S00. Then, the hok/sok system was introduced into the plasmid ptrc99a-ectABC of strain S03, obtaining strain S04. Without antibiotics addition, the ectoine titer of S04 harboring ptrc99a-ectABC-hok reached 4.1 g/L, which was 36.7% higher than that of S03 (Fig. 7B). In order to evaluate the stability and ectoine production of the strains S04 and S03, the fed-batch fermentation was carried out in a 5 L fermenter. Finally, the ectoine titer of strain S04 reached 23.3 g/L, which was 1.5-fold higher than S03 (15.9 g/L). The growth of strains S03 and S04 showed different trend: the OD600 of S03 reached the maximum at 28 h and decreased at 36 h, so the accumulation rate of ectoine production of S03 slowed down at 28h; while the ectoine production of S04 continuously increased with growing biomass of S04. The productivity of S04 was 0.53 g/L/h, which was 1.2-fold higher than that of S03 (0.45 g/L/h) (Fig. 7C, 7D). It was indicated that the hok/sok system was also beneficial for improving plasmid stability and ectoine production, which also could eliminate the environmental concerns associated with the usage of antibiotics.