Optimizing Hexose Utilization Pathways of Cupriavidus necator for Improving Growth and L-Alanine Production under Heterotrophic and Autotrophic Conditions

Cupriavidus necator is a versatile microbial chassis to produce high-value products. Blocking the poly-β-hydroxybutyrate synthesis pathway (encoded by the phaC1AB1 operon) can effectively enhance the production of C. necator, but usually decreases cell density in the stationary phase. To address this problem, we modified the hexose utilization pathways of C. necator in this study by implementing strategies such as blocking the Entner–Doudoroff pathway, completing the phosphopentose pathway by expressing the gnd gene (encoding 6-phosphogluconate dehydrogenase), and completing the Embden–Meyerhof–Parnas pathway by expressing the pfkA gene (encoding 6-phosphofructokinase). During heterotrophic fermentation, the OD600 of the phaC1AB1-knockout strain increased by 44.8% with pfkA gene expression alone, and by 93.1% with gnd and pfkA genes expressing simultaneously. During autotrophic fermentation, gnd and pfkA genes raised the OD600 of phaC1AB1-knockout strains by 19.4% and 12.0%, respectively. To explore the effect of the pfkA gene on the production of C. necator, an alanine-producing C. necator was constructed by expressing the NADPH-dependent L-alanine dehydrogenase, alanine exporter, and knocking out the phaC1AB1 operon. The alanine-producing strain had maximum alanine titer and yield of 784 mg/L and 11.0%, respectively. And these values were significantly improved to 998 mg/L and 13.4% by expressing the pfkA gene. The results indicate that completing the Embden–Meyerhof–Parnas pathway by expressing the pfkA gene is an effective method to improve the growth and production of C. necator.


Introduction
Cupriavidus necator H16 (formerly known as Ralstonia eutropha H16) is a chemolithoautotrophic Gram-negative bacterium that serves as a model organism for the natural synthesis of poly-β-hydroxybutyrate (PHB).It has been shown to accumulate up to 90% of PHB based on dry weight under restricted nutrition [1].Its broad carbon source utilization spectrum, detailed genomic information, and efficient gene editing system [2,3] position C. necator as a promising candidate for applications in environmental protection and bio-resource conversion [4].Studies have shown that C. necator can utilize waste oil, lignocellulose-derived sugar, and inedible rice to produce PHB [5][6][7].Additionally, several kinds of biofuels, like alcohol [8] and fatty acids [9], have also been successfully synthesized in C. necator by metabolic engineering.With H 2 providing reducing power and O 2 acting as the electron acceptor, C. necator fixed CO 2 through the Calvin-Benson-Basham cycle and flowed it into the central carbon metabolic pathway [10].Recent studies have modified C. necator to convert CO 2 into high-value products like glucose, acetoin, and 2-hydroxyisobutyrate [11][12][13].These findings demonstrate that C. necator is a versatile microbial chassis for high-value biochemical production from CO 2 .
In nature, the Embden-Meyerhof-Parnas (EMP) pathway and the pentose phosphate (PP) pathway are the main central carbon metabolism systems for most microbes.These pathways convert glucose into pyruvate while providing essential energy and precursors to maintain the basic metabolisms, such as DNA replication and protein synthesis.Generally, the EMP pathway collaborates with the PP pathway to regulate energy balance and coenzyme supply [14].However, the 6-phosphofructokinase (encoded by the pfkA gene) in the EMP pathway and 6-phosphogluconate dehydrogenase (encoded by the gnd gene) in the PP pathway are absent in C. necator [15], indicating that hexose is mainly metabolized through the Entner-Doudoroff (ED) pathway during heterotrophic fermentation (Figure 1).Compared with the EMP pathway, the ED pathway offers a shorter and faster metabolic process and provides additional NADPH, although it has a lower adenosine triphosphate (ATP) supply.The glyceraldehyde 3-phosphate generated in the ED pathway not only follows the downstream of the EMP pathway but also participates in the PP pathway (Figure 1) to generate ribose 5-phosphate and erythrose 4-phosphate, which serve as precursors for nucleic acid and phospholipid syntheses, respectively.Consequently, it is hypothesized that the incomplete EMP and PP pathways in C. necator may inhibit cell growth during fermentation.
In nature, the Embden-Meyerhof-Parnas (EMP) pathway and the pentose phosphate (PP) pathway are the main central carbon metabolism systems for most microbes.These pathways convert glucose into pyruvate while providing essential energy and precursors to maintain the basic metabolisms, such as DNA replication and protein synthesis.Generally, the EMP pathway collaborates with the PP pathway to regulate energy balance and coenzyme supply [14].However, the 6-phosphofructokinase (encoded by the pfkA gene) in the EMP pathway and 6-phosphogluconate dehydrogenase (encoded by the gnd gene) in the PP pathway are absent in C. necator [15], indicating that hexose is mainly metabolized through the Entner-Doudoroff (ED) pathway during heterotrophic fermentation (Figure 1).Compared with the EMP pathway, the ED pathway offers a shorter and faster metabolic process and provides additional NADPH, although it has a lower adenosine triphosphate (ATP) supply.The glyceraldehyde 3-phosphate generated in the ED pathway not only follows the downstream of the EMP pathway but also participates in the PP pathway (Figure 1) to generate ribose 5-phosphate and erythrose 4-phosphate, which serve as precursors for nucleic acid and phospholipid syntheses, respectively.Consequently, it is hypothesized that the incomplete EMP and PP pathways in C. necator may inhibit cell growth during fermentation.Under the regulation of the phaC1AB1 operon (encoding three genes catalyzing the generation of PHB from acetyl-CoA), C. necator consumes acetyl-CoA as precursor and NADPH as coenzyme to form PHB, which accumulates in cells as the main energy storage material [16,17].During metabolic engineering, knocking out the phaC1AB1 operon is common practice to divert sufficient carbon flux towards product synthesis.However, this approach often leads to a significant reduction in cell density, since the knockout of the phaC1AB1 operon may cause metabolic disorders of carbon flux, energy supply, and Under the regulation of the phaC1AB1 operon (encoding three genes catalyzing the generation of PHB from acetyl-CoA), C. necator consumes acetyl-CoA as precursor and NADPH as coenzyme to form PHB, which accumulates in cells as the main energy storage material [16,17].During metabolic engineering, knocking out the phaC1AB1 operon is common practice to divert sufficient carbon flux towards product synthesis.However, this approach often leads to a significant reduction in cell density, since the knockout of the phaC1AB1 operon may cause metabolic disorders of carbon flux, energy supply, and coenzyme balance [18].Noting that all these factors are involved in different glycolytic pathways, this study first explored the effect of regulating hexose utilization pathways on the growth of PHB-knockout C. necator.
L-alanine is an aliphatic amino acid with an annual global demand of approximately 50,000 tons [19].L-alanine is the only amino acid with a sweet taste and can be used as a sweetener, freshener, and seasoning agent in food [20,21].As a glucogenic amino acid, L-alanine plays an important role in the treatment of diabetes and related research [22].The catalytic synthesis of L-alanine from pyruvate by alanine dehydrogenase (encoded by the alaD gene) is a good choice for industrial production.As a model organism for PHB synthesis, C. necator has the ability to generate a large number of pyruvates, which provides sufficient substrates for L-alanine synthesis.The synthesis of L-alanine is directly influenced by pyruvate supply and coenzyme balance (Figure 1).Therefore, an alanine-producing C. necator was constructed in this study to validate the effect of hexose utilization pathways on alanine production.The purpose of this study was to develop a regulating strategy for C. necator in enhancing the production of high-value products based on pyruvate, relieving growth inhibition caused by phaC1AB1 operon knockout, and laying the foundation for C. necator low-carbon industrial production through autotrophic fermentation.

Effect of Blocking the ED and PHB Synthesis Pathways on Growth
To avoid potential interference with foreign gene expression, the H16_A0006 gene, encoding the R subunit of type I restriction enzyme, was first knocked out in the wild-type C. necator to obtain strain Cn∆6.Strain Cn∆6∆PHB was easily obtained by knocking out the phaC1AB1 operon of strain Cn∆6.To block the ED pathway (Figure 1), attempts were made to delete or replace the eda and edd genes (encoding 2-keto-3-deoxy-6-phosphogluconate aldolase and phosphogluconate dehydratase, respectively) with the gnd or pfkA genes, but all attempts failed.Instead, we selected the strain Cn∆6∆zwf (knocking out the zwf1, zwf2, and zwf3 genes in Cn∆6, encoding glucose-6-phosphate 1-dehydrogenase) as the ED-pathway-blocked strain and successfully knocked out the PHB synthesis pathway, obtaining the strain Cn∆6∆zwf∆PHB.
In the heterotrophic fermentation supplied with 30 g/L fructose as the sole carbon source, the growth rate of C. necator decreased notably after the knockout of zwf genes, and the duration of the logarithmic phase was extended from 6 to 10 days.Meanwhile, the cell density visibly decreased from 22.7 to 4.1 (Figure 2A).In contrast, zwf genes had no substantial impact on the growth or cell density of C. necator in autotrophic fermentation (Figure 2B), probably because the zwf genes were not involved in the carbon flux during autotrophic fermentation.In the stationary phase, the knockout of the phaC1AB1 operon obviously reduced the cell density of Cn∆6 both in heterotrophic (from 22.7 to 6.0) and autotrophic (from 6.1 to 3.8) fermentation.In addition, no interaction was observed between the zwf genes and phaC1AB1 operon affecting the growth of C. necator in either heterotrophic or autotrophic fermentation.

Effect of Completing the EMP and PP Pathways on Growth
The EMP and PP pathways are incomplete in C. necator for the lack of 6-phosphofructok inase and 6-phosphogluconate dehydrogenase, respectively.Here, we successfully constructed pBBR1-series plasmids (Table 1) that overexpressed the gnd and pfkA genes (both cloned from Escherichia coli) and electrically transformed them into ED and PHB synthesis pathway-blocking strains.Since the empty plasmid pBBR1-MCS2 had no effect on the growth of C. necator (Figure S1), all the differences in growth were attributed to the changes in hexose utilization pathways.
no substantial impact on the growth or cell density of C. necator in autotrophic fermentation (Figure 2B), probably because the zwf genes were not involved in the carbon flux during autotrophic fermentation.In the stationary phase, the knockout of the phaC1AB1 operon obviously reduced the cell density of CnΔ6 both in heterotrophic (from 22.7 to 6.0) and autotrophic (from 6.1 to 3.8) fermentation.In addition, no interaction was observed between the zwf genes and phaC1AB1 operon affecting the growth of C. necator in either heterotrophic or autotrophic fermentation.In the heterotrophic fermentation supplemented with 30 g/L fructose, the lag phases of Cn∆6, Cn∆6∆PHB, and Cn∆6∆zwf∆PHB were prolonged remarkably by expressing the gnd gene (Figure 3A-C).The pfkA gene conspicuously increased the cell densities of Cn∆6∆PHB (from 6.1 to 7.2 in Figure 3B) and Cn∆6∆zwf∆PHB (from 2.9 to 4.2 in Figure 3C).Overexpression of both the gnd and pfkA genes inhibited the growth of Cn∆6 (from 22.7 to 17.0 in Figure 3A).Conversely, the cell density in the stationary phase was apparently increased from 6.1 to 6.8 in Cn∆6∆PHB-gndpfkA (Figure 3B) and from 2.9 to 5.6 in Cn∆6∆zwf∆PHB-gndpfkA (Figure 3C).While in autotrophic fermentation, expression of the gnd gene no longer caused growth delay.Both the gnd and pfkA genes markedly increased the highest cell density of Cn∆6 by 18.0% and 21.3%, respectively (Figure 3D).This effect persisted in Cn∆6∆PHB with increases of 19.4% and 12.0%, respectively (Figure 3E).After knocking out the zwf genes, Gnd lost its biofunction due to the lack of substrate.Therefore, only the pfkA gene was overexpressed to complete the EMP pathway in strain Cn∆6∆zwf∆PHB, but no noteworthy effect on strain growth was observed (Figure 3F).).Overexpression of both the gnd and pfkA genes inhibited the growth of CnΔ6 (from 22.7 to 17.0 in Figure 3A).Conversely, the cell density in the stationary phase was apparently increased from 6.1 to 6.8 in CnΔ6ΔPHB-gndpfkA (Figure 3B) and from 2.9 to 5.6 in CnΔ6ΔzwfΔPHB-gndpfkA (Figure 3C).While in autotrophic fermentation, expression of the gnd gene no longer caused growth delay.Both the gnd and pfkA genes markedly increased the highest cell density of CnΔ6 by 18.0% and 21.3%, respectively (Figure 3D).This effect persisted in CnΔ6ΔPHB with increases of 19.4% and 12.0%, respectively (Figure 3E).After knocking out the zwf genes, Gnd lost its biofunction due to the lack of substrate.Therefore, only the pfkA gene was overexpressed to complete the EMP pathway in strain CnΔ6ΔzwfΔPHB, but no noteworthy effect on strain growth was observed (Figure 3F).

Construction of the Alanine-Producing Strain
Compared with fructose, glucose is cheaper and more suitable for industrial-scale production.Previous studies have reported that glucose can be utilized by C. necator through two approaches: expressing the exogenous glf gene, encoding an energy-independent glucose-facilitated diffusion transporter [23], or modifying the native N-acetylglucosamine transport system by mutation of NagE(G265R) and deletion of the nagR gene [24].After construction and verification (Figure S2), the second strategy showed better growth and was applied in strain CnΔ6 for the production of alanine, resulting in strain CnΔ6RE.The alaE gene (encoding an alanine exporter in E. coli) was fused with the PphaC1 promoter and inserted into the ldhA1A2 locus of CnΔ6RE, obtaining strain CnΔ6REalaEΔldhA12, thereby promoting the export of alanine as well as blocking the synthesis of by-product lactate.Subsequently, the phaC1AB1 operon in the genome of CnΔ6REalaEΔldhA12 was knocked out to obtain the strain CnAla.
Using NADH as a coenzyme, L-alanine dehydrogenase (encoded by the alaD gene) from G. stearothermophilus [25] and L. sphaericus [26] has been reported for alanine

Construction of the Alanine-Producing Strain
Compared with fructose, glucose is cheaper and more suitable for industrial-scale production.Previous studies have reported that glucose can be utilized by C. necator through two approaches: expressing the exogenous glf gene, encoding an energy-independent glucose-facilitated diffusion transporter [23], or modifying the native N-acetylglucosamine transport system by mutation of NagE(G265R) and deletion of the nagR gene [24].After construction and verification (Figure S2), the second strategy showed better growth and was applied in strain Cn∆6 for the production of alanine, resulting in strain Cn∆6RE.The alaE gene (encoding an alanine exporter in E. coli) was fused with the P phaC1 promoter and inserted into the ldhA1A2 locus of Cn∆6RE, obtaining strain Cn∆6REalaE∆ldhA12, thereby promoting the export of alanine as well as blocking the synthesis of by-product lactate.Subsequently, the phaC1AB1 operon in the genome of Cn∆6REalaE∆ldhA12 was knocked out to obtain the strain CnAla.
Using NADH as a coenzyme, L-alanine dehydrogenase (encoded by the alaD gene) from G. stearothermophilus [25] and L. sphaericus [26] has been reported for alanine production.Scientists have engineered the alaD gene from B. subtilis to change its coenzyme preference to NADPH [27].The above three alaD genes were codon-optimized and expressed in plasmids with different promoters for alanine synthesis in this study.During heterotrophic fermentation supplied with 30 g/L glucose, no alanine was detected in the strain Cn∆6REalaE∆ldhA12, whether expressing any alaD gene with P phaC1 or araCP BAD promoter.Following the knockout of the phaC1AB1 operon, alanine was significantly accumulated in strain CnAla (Figure 4).The constitutive and inducible expression of the alaD gs gene in strain CnAla exhibited similar alanine production at approximately 249 mg/L.However, the alaD ls gene was not suitable for alanine synthesis in C. necator.Compared with 356 mg/L of alanine titer in constitutive expression, the inducible expression of the alaD NADPH gene in strain CnAla-araPalaD NADPH (CnAlaD) showed the highest alanine titer at 784 mg/L.Furthermore, autotrophic fermentation of alanine-producing strains was also conducted in CnMM using CO 2 as the sole carbon source, but no alanine accumulation was observed.This could be attributed to the absence of an organic carbon source in CnMM, leading to the consumption of synthesized alanine before it could be transported into the culture medium.
production.Scientists have engineered the alaD gene from B. subtilis to change its c zyme preference to NADPH [27].The above three alaD genes were codon-optimized expressed in plasmids with different promoters for alanine synthesis in this study.Du heterotrophic fermentation supplied with 30 g/L glucose, no alanine was detected in strain CnΔ6REalaEΔldhA12, whether expressing any alaD gene with PphaC1 or araC promoter.Following the knockout of the phaC1AB1 operon, alanine was significantly a mulated in strain CnAla (Figure 4).The constitutive and inducible expression of the al gene in strain CnAla exhibited similar alanine production at approximately 249 mg/L.H ever, the alaDls gene was not suitable for alanine synthesis in C. necator.Compared with mg/L of alanine titer in constitutive expression, the inducible expression of the alaDN gene in strain CnAla-araPalaDNADPH (CnAlaD) showed the highest alanine titer at 784 m Furthermore, autotrophic fermentation of alanine-producing strains was also conducte CnMM using CO2 as the sole carbon source, but no alanine accumulation was obser This could be attributed to the absence of an organic carbon source in CnMM, leading to consumption of synthesized alanine before it could be transported into the culture med

Effect of Completing the EMP and PP Pathways on Alanine Production
To complete the EMP and PP pathways, the gnd and pfkA genes were separately pressed in strain CnAlaD downstream of the alaD gene in plasmids, along with the P promoter.Consistent with previous results, the expression of the gnd gene prolonged lag period of strain CnAlaD from 1 d to 3 d without changing the highest cell den (Figure 5A).In addition, expressing the gnd gene also caused a significant decrease in nine titer (from 784 mg/L to 186 mg/L, Figure 5B) and yield (from 11.0% to 1.6%, Fig 5B).In contrast, the expression of the pfkA gene markedly enhanced the maximum tit alanine from 784 mg/L to 998 mg/L, and the yield of glucose converted to alanine creased from 11.0% to 13.4% (Figure 5B).The extended lag phase and decreased ala production caused by the gnd gene were partially mitigated when it was co-expre with the pfkA gene.Effect of different alaD genes on the growth and alanine titer of strain CnAla.The highest OD 600 and alanine titer of the strain were observed with the constitutive (by P phaC1 , marked with P) or inducible (by araCP BAD , marked with araP) expression of different alaD genes in plasmids during heterotrophic fermentation with 30 g/L glucose as the sole carbon source.The alaD gs , alaD ls , and alaD NADPH genes were codon-optimized from G. stearothermophilus, L. sphaericus, and B. subtilis, respectively.

Effect of Completing the EMP and PP Pathways on Alanine Production
To complete the EMP and PP pathways, the gnd and pfkA genes were separately expressed in strain CnAlaD downstream of the alaD gene in plasmids, along with the P phaC1 promoter.Consistent with previous results, the expression of the gnd gene prolonged the lag period of strain CnAlaD from 1 d to 3 d without changing the highest cell density (Figure 5A).In addition, expressing the gnd gene also caused a significant decrease in alanine titer (from 784 mg/L to 186 mg/L, Figure 5B) and yield (from 11.0% to 1.6%, Figure 5B).In contrast, the expression of the pfkA gene markedly enhanced the maximum titer of alanine from 784 mg/L to 998 mg/L, and the yield of glucose converted to alanine increased from 11.0% to 13.4% (Figure 5B).The extended lag phase and decreased alanine production caused by the gnd gene were partially mitigated when it was co-expressed with the pfkA gene.

Discussion
In line with previous reports [9,16], the present study found that knocking out the phaC1AB1 operon reduced the cell density of C. necator both in heterotrophic and autotrophic fermentation.This result contrasts with the findings of Lu et al. [28], who reported minimal impact on the growth in heterotrophic fermentation with the deletion of the phaC1AB1 operon.Wang et al. [29] observed a decrease in cell density in the PHB-knockout strain using glucose as the sole carbon source, but this effect disappeared when glycerol replaced glucose.These results suggest that the carbon source might be a factor that changes the effect of the phaC1AB1 operon knockout on the cell density of C. necator.Gluconate, fructose, and N-acetylglucosamine are the only three carbohydrates utilized by unmodified C. necator.Additionally, several different types of carbon sources, such as acetate, citrate, and glycerol, for instance, can also be utilized by C. necator [30].Under unbalanced culture conditions, like limitation of oxygen, phosphorus, or nitrogen, a large amount of PHB was accumulated in cells of C. necator as energy storage material [1].The mineral media for C. necator were optimized in this study, especially increasing the nitrogen concentration and the proportion of trace elements, which accelerated the growth rate of the CnΔ6 strain and significantly shortened the logarithmic phase of autotrophic fermentation from 64 d to 15 d [11].These findings imply that various carbon sources and C/N ratios in the medium may also affect the growth of PHB-knockout strains and this merits further investigation in future studies.
The expression of the gnd gene partly directed the carbon flux towards the PP pathway, accompanied by the generation of a large amount of NADPH.The blocking of the PHB synthesis pathway further reduced NADPH consumption.The excess NADPH, functioning as a reductant, might induce oxidative stress in cells [31] and further manifest as growth delay or lower production property.Based on KEGG annotations [32], the cbbc operon in the chromosome and the cbbp operon in megaplasmid pHG1 were expressed independently, both capable of achieving CO2 fixation using NADPH as a coenzyme [33,34].The balanced NADPH may explain why the extended lag phase caused by gnd gene expression only occurs in heterotrophic fermentation.Similarly, the expression of the NADPH-dependent alaD gene had higher alanine production, as it alleviated the stress associated with coenzyme imbalance.Apart from the imbalanced coenzyme, it has been reported that the overexpression of the gnd gene also reduced the intracellular acetyl-CoA concentration of C. necator [35], resulting in insufficient energy supply and reflected as lower alanine production in strain CnAlaD-gnd.In consequence, completing the PP pathway by expressing the gnd gene is not conducive to the biosynthetic modifications of C. necator.

Discussion
In line with previous reports [9,16], the present study found that knocking out the phaC1AB1 operon reduced the cell density of C. necator both in heterotrophic and autotrophic fermentation.This result contrasts with the findings of Lu et al. [28], who reported minimal impact on the growth in heterotrophic fermentation with the deletion of the phaC1AB1 operon.Wang et al. [29] observed a decrease in cell density in the PHBknockout strain using glucose as the sole carbon source, but this effect disappeared when glycerol replaced glucose.These results suggest that the carbon source might be a factor that changes the effect of the phaC1AB1 operon knockout on the cell density of C. necator.Gluconate, fructose, and N-acetylglucosamine are the only three carbohydrates utilized by unmodified C. necator.Additionally, several different types of carbon sources, such as acetate, citrate, and glycerol, for instance, can also be utilized by C. necator [30].Under unbalanced culture conditions, like limitation of oxygen, phosphorus, or nitrogen, a large amount of PHB was accumulated in cells of C. necator as energy storage material [1].The mineral media for C. necator were optimized in this study, especially increasing the nitrogen concentration and the proportion of trace elements, which accelerated the growth rate of the Cn∆6 strain and significantly shortened the logarithmic phase of autotrophic fermentation from 64 d to 15 d [11].These findings imply that various carbon sources and C/N ratios in the medium may also affect the growth of PHB-knockout strains and this merits further investigation in future studies.
The expression of the gnd gene partly directed the carbon flux towards the PP pathway, accompanied by the generation of a large amount of NADPH.The blocking of the PHB synthesis pathway further reduced NADPH consumption.The excess NADPH, functioning as a reductant, might induce oxidative stress in cells [31] and further manifest as growth delay or lower production property.Based on KEGG annotations [32], the cbb c operon in the chromosome and the cbb p operon in megaplasmid pHG1 were expressed independently, both capable of achieving CO 2 fixation using NADPH as a coenzyme [33,34].The balanced NADPH may explain why the extended lag phase caused by gnd gene expression only occurs in heterotrophic fermentation.Similarly, the expression of the NADPH-dependent alaD gene had higher alanine production, as it alleviated the stress associated with coenzyme imbalance.Apart from the imbalanced coenzyme, it has been reported that the overexpression of the gnd gene also reduced the intracellular acetyl-CoA concentration of C. necator [35], resulting in insufficient energy supply and reflected as lower alanine production in strain CnAlaD-gnd.In consequence, completing the PP pathway by expressing the gnd gene is not conducive to the biosynthetic modifications of C. necator.
In the present study, the pfkA gene significantly improved the cell density, alanine titer, and yield of C. necator.These results suggested that completing the EMP pathway by expressing the pfkA gene is able to boost glucose utilization efficiency in C. necator.The expression of the pfkA gene enabled the bidirectional flow of carbon flux, which originally flowed unidirectionally into the PP pathway, thereby achieving dynamic regulation of intracellular ribose (used for DNA synthesis) and pyruvate (supplied energy and carbon skeleton).In addition, the E. coli pfkA was found to realize the fructose oxidative decomposition through the EMP instead of the ED pathway [36].The ATP throughput of the EMP pathway is higher than that of the ED pathway.Accordingly, it is essential to consider the ATP balance when strengthening the EMP pathway by overexpressing the pfkA gene.Cell growth and L-serine accumulation were improved after pfkA was over-expressed in Corynebacterium glutamicum [37].The expression of pfkA in Pseudomonas putida inhibited cell growth, decreased ATP and NADPH in cells, and increased sensitivity to oxidative stress [38].Conversely, the ATP and butanol production in Clostridium acetobutylicum were enhanced by overexpressing pfkA [39].Hence, the appropriate pfkA gene expression level is crucial to demonstrate the advantage of ATP supply in the EMP pathway, ultimately resulting in faster cell growth and increased production.
The core strategy was to increase alanine production by improving the carbon flow of the alanine synthesis pathway.In terms of precursor supply, strengthening the EMP and ED pathways has great potential to increase the hexose utilization ability and enhance the supply of pyruvate [40].Following the same rule, the expression of the pfkA gene increased the efficiency of pyruvate supply, thereby increasing the production of alanine in C. necator.In addition to precursor supply, screening for alanine dehydrogenase suitable for the chassis microbe can also redirect more carbon flow toward alanine synthesis [41].As shown in the results of this study, NADPH-dependent alanine dehydrogenase derived from B. subtilis exhibited the highest alanine synthesis ability in C. necator when expressed with the strong arabinose-induced promoter.
In conclusion, an alanine-producing strain CnAlaD was constructed for the first time in the present study, demonstrating that C. necator is a valuable chassis organism for amino acid biosynthesis.Moreover, completing the EMP pathway by pfkA gene expression prominently improved cell density, alanine titer, and yield, which proves that the regulation of hexose utilization pathways is a powerful method to increase the production of C. necator, wherein the ATP balance and coenzyme balance need further exploration in future studies.

Strains and Culture Medium
All the strains involved in this study are shown in Table 2.The Escherichia coli Trans1-T1 (Transgen, Beijing, China) and S17-1 were adopted for plasmid construction and conjugation, respectively, cultivated in Luria-Bertani (LB) medium at 37 • C. Another 10 g/L fructose, as the optimal carbon source, was added to LB for the cultivation of C. necator at 30 • C. When solid culture was required, an additional 2% of agar was supplied to the medium.The mineral medium for C. necator (CnMM) was optimized from a previous study [42], containing 7 g/L Na 2 HPO 4 , 3 g/L KH 2 PO 4 , 5 g/L (NH 4 ) 2 SO 4 , 0.3 g/L NaHCO 3 , 0.2 g/L MgSO 4 •7H 2 O, 0.001 g/L CaSO 4 , and 3 mL/L trace element.The trace element was redesigned according to previous studies [43][44][45], including 5 g/L ferric citrate, 1 g/L CaCl 2 , 0.6 g/L NiSO 4 •7H 2 O, 0.5 g/L ZnSO 4 •7H 2 O, 0.3 g/L CoCl 2 •6H 2 O, 0.3 g/L H 3 BO   All plasmids and primers designed for this study are listed in Table 1 and Table S1, respectively.The target gene fragments and promoters were obtained by PCR using primers with homologous arms, overlapped to the linearized pBBR1 plasmid (by EcoRI), and assembled using the Gibson assembly.For the genomic editing of C. necator by homologous recombination, the upstream and downstream homologous arms (about 600 bp) of the editing site were amplified and assembled to the linearized pK18mobsacB plasmid (by EcoRI and SmaI).All constructed plasmids were verified by sequencing before enrichment and electroporation.

Strain Modification
Transconjugation [8] was adopted in the plasmid transformation of C. neacator, where the recombinant plasmid of pK18mobsacB was electro-transformed to E. coli S17-1 first and then integrated into the genome of C. necator after 24 h of co-culture.The single-crossover colony of C. necator was screened using kanamycin and verified by PCR.A positive colony was then counter-selected in an LB solid plate with 50 g/L of sucrose [11].The double-crossover colony was identified by colony PCR and sequencing.
In order to produce alanine and complement the EMP and PP pathways, plasmids carrying alaD, pfkA, or gnd genes were transformed into electrocompetent cells from C. necator by electroporation in a 2 mm cuvette with 2300 V of voltage, 25 µF of capacitance, and 200 Ω of resistance.Transformed strains were spread on LB solid medium supplemented with kanamycin after 2 h cultivation in a 30 • C shaker at 200 r/min with 1 mL of LB, and then the colonies that appeared on the plate were verified by colony PCR.

Heterotrophic Fermentation
C. necator was heterotrophically cultivated in 100 mL shake flasks containing 50 mL of CnMM, supplying 30 g/L of fructose or glucose as the sole carbon source.Strains were enriched overnight in LB, centrifuged, washed with phosphate-buffered saline (pH = 7.4), and then inoculated into flasks with an initial OD 600 of 0.03.The heterotrophic fermentation was carried out in a shaker at 30 • C and 200 r/min.Cell density and alanine accumulation were measured every 24 h with 3 replicates per strain.

Autotrophic Fermentation
Autotrophic fermentation was performed in 250 mL anaerobic bottles, each containing 50 mL of CnMM.The residual gas in the anaerobic bottle was sufficiently replaced every 24 h with a fresh gas mixture (H 2 :CO 2 :O 2 = 8:1:1 in volume) at 1 L/min for 3 min, using the mixed gas charging system (Figure S3) to ensure the carbon and energy supply [29].The inoculation process was the same with heterotrophic fermentation, except for the initial OD 600 at 1.0.Autotrophic fermentation was also carried out in a 200 r/min shaker at 30 • C. Samples were taken every 5 d to determine cell density and alanine concentration with 3 replicates per strain.

Analytical Methods
For the detection of cell density, 200 µL of diluted bacterial solution was divided into 96-well plates and OD 600 was determined with a microplate reader (BioTek, Burlington, VT, USA).The composition of the fermentation broth was detected by HPLC.Glucose was eluted with 5 mmol/L of H 2 SO 4 at 0.5 mL/min at 35 • C using an Animex HPX-87H (9 µm, 7.8 × 300 mm) column (Bio-Rad, Hercules, CA, USA) and quantified using a refractive index detector.Alanine was analyzed using the AJS-01 Amino Acid Analysis Method Package (Shimadzu, Sakyoku, Kyoto, Japan).Specifically, alanine was first derived with o-phthalaldehyde online and then flowed into an AJS-02 (3 µm, 4.6 × 150 mm) column; subsequent gradient elution was performed using solvent A (4.5 g/L Na 2 HPO 4 •12H 2 O and 4.25 g/L Na 2 B 4 O 7 •10H 2 O, pH adjusted to 8.2 with HCl) and solvent B (water, methanol, and acetonitrile mixed at a volume ratio of 10:45:45) at a flow rate of 2 mL/min under a temperature of 50 • C; the compounds were finally identified using a UV-vis detector at 338 nm.The growth curve and alanine accumulation were summarized and analyzed with GraphPad Prism (v8.0.2,GraphPad Software, Boston, MA, USA), adopting a paired T-test or one-way analysis of variance to compare the significance of differences in two or more groups of data.

Figure 1 .
Figure 1.Hexose utilization pathways and metabolic engineering of C. necator for alanine production.The endogenous ED pathway was blocked by the knockout of glucose-6-phosphate 1-dehydrogenase (encoded by the zwf gene).The EMP pathway and PP pathway were completed by expressing the pfkA gene (encoding 6-phosphofructokinase) and gnd gene (encoding 6-phosphogluconate dehydrogenase) from E. coli, respectively.The phaC1AB1 operon was knocked out to increase the supply of precursors.The alaD gene (encoding L-alanine dehydrogenase) and alaE gene (encoding an alanine exporter in E. coli) were heterologously expressed for the synthesis and export of L-alanine.nagE, encoding a subunit of a putative GlcNAc-specific phosphotransferase system; nagR, encoding a putative GntRtype transcriptional regulator of nagE; edd, encoding phosphogluconate dehydratase; eda, encoding 2-keto-3-deoxy-6-phosphogluconate aldolase; ldhA1A2, encoding D-lactate dehydrogenase; phaA, encoding acetyl-CoA acetyltransferase; phaB, encoding acetoacetyl-CoA reductase; phaC, encoding poly(3-hydroxybutyrate) polymerase.

Figure 2 .
Figure 2. Effect of the ED and PHB synthesis pathways on the growth of C. necator.All of the zwf1, zwf2, and zwf3 genes were knocked out in both Cn∆6∆zwf and Cn∆6∆zwf∆PHB.The phaC1AB1 operon was knocked out in both Cn∆6∆PHB and Cn∆6∆zwf∆PHB.(A) The growth curves of C. necator during heterotrophic fermentation with 30 g/L fructose as the sole carbon source.(B) The growth curves of C. necator during autotrophic fermentation with CO 2 as the sole carbon source.

Figure 3 .
Figure 3.Effect of the pfkA and gnd genes on the growth of different C. necator strains.Both the pfkA and gnd genes were constitutively expressed with promoter PphaC1 in plasmids.Heterotrophic fermentation using 30 g/L fructose as the sole carbon source and autotrophic fermentation using CO2 as the sole carbon source.Both the pfkA and gnd genes were cloned from E. coli.(A) The changes in the growth of CnΔ6 during heterotrophic fermentation; (B) the changes in the growth of CnΔ6ΔPHB during heterotrophic fermentation; (C) the changes in the growth of CnΔ6ΔzwfΔPHB during heterotrophic fermentation; (D) the changes in the growth of CnΔ6 during autotrophic fermentation; (E) the changes in the growth of CnΔ6ΔPHB during autotrophic fermentation; (F) the changes in the growth of CnΔ6ΔzwfΔPHB during autotrophic fermentation.

Figure 3 .
Figure 3.Effect of the pfkA and gnd genes on the growth of different C. necator strains.Both the pfkA and gnd genes were constitutively expressed with promoter P phaC1 in plasmids.Heterotrophic fermentation using 30 g/L fructose as the sole carbon source and autotrophic fermentation using CO 2 as the sole carbon source.Both the pfkA and gnd genes were cloned from E. coli.(A) The changes in the growth of Cn∆6 during heterotrophic fermentation; (B) the changes in the growth of Cn∆6∆PHB during heterotrophic fermentation; (C) the changes in the growth of Cn∆6∆zwf∆PHB during heterotrophic fermentation; (D) the changes in the growth of Cn∆6 during autotrophic fermentation; (E) the changes in the growth of Cn∆6∆PHB during autotrophic fermentation; (F) the changes in the growth of Cn∆6∆zwf∆PHB during autotrophic fermentation.

Figure 4 .
Figure 4. Effect of different alaD genes on the growth and alanine titer of strain CnAla.The hig OD600 and alanine titer of the strain were observed with the constitutive (by PphaC1, marked wi or inducible (by araCPBAD, marked with araP) expression of different alaD genes in plasmids du heterotrophic fermentation with 30 g/L glucose as the sole carbon source.The alaDgs, alaDls, alaDNADPH genes were codon-optimized from G. stearothermophilus, L. sphaericus, and B. subtili spectively.

Figure 4 .
Figure 4.Effect of different alaD genes on the growth and alanine titer of strain CnAla.The highest OD 600 and alanine titer of the strain were observed with the constitutive (by P phaC1 , marked with P) or inducible (by araCP BAD , marked with araP) expression of different alaD genes in plasmids during heterotrophic fermentation with 30 g/L glucose as the sole carbon source.The alaD gs , alaD ls , and alaD NADPH genes were codon-optimized from G. stearothermophilus, L. sphaericus, and B. subtilis, respectively.

Figure 5 .
Figure 5.Effect of the expression of the gnd and pfkA genes on the growth and the alanine production of strain CnAlaD.Both the gnd and pfkA genes were expressed in plasmids.Heterotrophic fermentation using 30 g/L glucose as the sole carbon source.Arrows represent the time point to add inducer.(A) The growth curves of strain CnAlaD with different plasmids.(B) The highest titer and yield of glucose converted to alanine of strain CnAlaD with different plasmids.

Figure 5 .
Figure 5.Effect of the expression of the gnd and pfkA genes on the growth and the alanine production of strain CnAlaD.Both the gnd and pfkA genes were expressed in plasmids.Heterotrophic fermentation using 30 g/L glucose as the sole carbon source.Arrows represent the time point to add inducer.(A) The growth curves of strain CnAlaD with different plasmids.(B) The highest titer and yield of glucose converted to alanine of strain CnAlaD with different plasmids.

Table 1 .
Plasmids used in this study.
pK18mobsacB-derived, for mutation of nagE(G793C) This study pK18-alaE-ldhA1A2 pK18mobsacB-derived, for the replacement of ldhA1A2 with alaE from E. coli fused with P phaC1 This study pBBR1-MCS2 Wide host plasmid for gene expression, with Cpa fdx terminator at the end of MCS, Km r Laboratory gnd-pBBR1 gnd gene from E. coli fused with P phaC1 and assembled into pBBR1-MCS2 This study pfkA-pBBR1 pfkA gene from E. coli fused with P phaC1 and assembled into pBBR1-MCS2 This study gnd-pfkA-pBBR1 pfkA gene from E. coli fused with P phaC1 and assembled after the gnd gene of gnd-pBBR1 This study alaD gs -pBBR1 Codon-optimized alaD gene from Geobacillus stearothermophilus fused with P phaC1 and assembled into pBBR1-MCS2 This study alaD ls -pBBR1 Codon-optimized alaD gene from Lysinibacillus sphaericus fused with P phaC1 and assembled into pBBR1-MCS2 This study alaD NADPH -pBBR1 Codon-optimized alaD gene from Bacillus subtilis, mutated to NADPH preference, fused with P phaC1 , and assembled into pBBR1-MCS2 This study araPalaD gs -pBBR1 Codon-optimized alaD gene from G. stearothermophilus fused with araCP 3 , 0.22 g/L MnSO 4 •H 2 O, 0.2 g/L Na 2 MoO 4 •2H 2 O, and 0.2 g/L CuSO 4 •5H 2 O.A sole carbon source, like fructose, glucose, or CO 2 , was added into CnMM to support the growth of strains.Kanamycin (50 µg/mL for E. coli, 200 µg/mL for C. necator) and gentamicin (10 µg/mL for C. necator) were added to the medium if necessary.

Table 2 .
Bacterial strains used in this study.