Increasing 1,4-Diaminobutane Production in Escherichia coli by Optimization of Cofactor PLP and NADPH Synthesis

1,4-diaminobutane is widely used in the industrial production of polymers, pharmaceuticals, agrochemicals and surfactants. Owing to economic and environmental concerns, there has been a growing interest in using microbes to produce 1,4-diaminobutane. However, there is lack of research on the influence of cofactors pyridoxal phosphate (PLP) and NADPH on the synthesis of 1,4-diaminobutane. PLP serves as a cofactor of ornithine decarboxylase in the synthesis of 1,4-diaminobutane. Additionally, the synthesis of 1 mol 1,4-diaminobutane requires 2 mol NADPH, thus necessitating consideration of NADPH balance in the efficient synthesis of 1,4-diaminobutane by Escherichia coli. The aim of this study was to enhance the synthesis efficiency of 1,4-diaminobutane through increasing production of PLP and NADPH. By optimizing the expression of the genes associated with synthesis of PLP and NADPH in E. coli, cellular PLP and NADPH levels increased, and the yield of 1,4-diaminobutane also increased accordingly. Ultimately, using glucose as the primary carbon source, the yield of 1,4-diaminobutane in the recombinant strain NAP19 reached 272 mg/L·DCW, by increased 79% compared with its chassis strain.


Introduction
The compound 1,4-diaminobutane, also known as putrescine, is not only involved in cellular metabolism and growth [1][2][3], but also a monomer material used in industry to produce PA46, PA410 and so on [4].The market demand for 1,4-diaminobutane is growing rapidly due to its high industrial value.At present, 1,4-diaminobutane is mainly produced from petroleum.Industrial scale production of 1,4-diaminbutane mainly relies on chemical synthesis by the addition of hydrogen cyanide to acrylanitrile to obtain succinonitrile, and then hydrogenation of succinonitrile under harsh reaction conditions.The process requires non-renewable petrochemicals as raw materials, as well as expensive catalysts, which can put pressure on environmental protection [5,6].Therefore, it is necessary to develop a new green production process for 1,4-diaminobutane.By constructing highyield 1,4-diaminobutane engineered strains, production of 1,4-diaminobutane via microbial fermentation has environmental advantages and represents the future direction of its industrialization.
Engineered strains for the production of 1,4-diaminobutane have been developed and made some progress.At present, the engineered strains were constructed generally by increasing the expression levels of genes in the 1,4-diaminobutane synthesis pathway [7][8][9], knocking out branching metabolic pathways and the degradation pathways of 1,4-diaminobutane [10,11], and screening for high-activity ornithine decarboxylase (ODC) to enhance the synthesis flux of 1,4-diaminobutane [12,13].In our laboratory, we

Optimisation of NADPH Synthesis in the 1,4-Diaminobutane Synthetic Pathway
In the 1,4-diaminobutane synthesis pathway, 2 mol NADPH is required to synthesize 1,4-diaminobutane.PntAB and PpnK catalyze NADP + and NADH to generate NADPH.In addition, overexpression of PP pathway could effectively increase NADPH concentration in strains.Therefore, in this study, the key genes involved in NADPH synthesis, pntAB and ppnK, as well as the genes in the PP pathway, were overexpressed to increase the production of NADPH and thus increased the production of 1,4-diaminobutane (Figure 1).

Optimisation of NADPH Synthesis in the 1,4-Diaminobutane Synthetic Pathway
In the 1,4-diaminobutane synthesis pathway, 2 mol NADPH is required to synthesize 1,4-diaminobutane.PntAB and PpnK catalyze NADP + and NADH to generate NADPH.In addition, overexpression of PP pathway could effectively increase NADPH concentration in strains.Therefore, in this study, the key genes involved in NADPH synthesis, pntAB and ppnK, as well as the genes in the PP pathway, were overexpressed to increase the production of NADPH and thus increased the production of 1,4-diaminobutane (Figure 1).

Overexpression of ppnK and pntAB Increased NADPH Supply
The cloned ppnK and pntAB genes were ligated into pETM6 plasmid and transformed into E. coli BL21(DE3) to form recombinant strains NAP1 and NAP2.Recombinant strains NAP1 and NAP2 could catalyze NADP + to NADPH by expressing pntAB and ppnK.NADPH synthesis and 1,4-diaminobutane yield were shown in Figure 2. The NADPH yield of NAP1 and NAP2 was significantly higher than that of the control strain E. coli BL21(DE3), and similarly, the yield of 1,4-diaminobutane was also increased companied with the elevation of NADPH, showing that improvement of NADPH can enhance the synthesis of 1,4-diaminobutane.The titer of 1,4-diaminobutane in NAP1 and NAP2 was 77 mg/L•DCW and 83 mg/L•DCW, respectively, which was about 27% and 37% higher than that in the control strain E. coli BL21(DE3).In order to furtherly increase the synthesis of NADPH, co-expression plasmid of pntAB and ppnK was transformed into E. coli BL21(DE3) to generate recombinant strain NAP3.The NADPH yield of NAP3 strain was higher, and the 1,4-diaminobutane titer was 95 mg/L•DCW, which was 57% greater than the control strain E. coli BL21(DE3) (Figure 2), indicating that the synthesis of 1,4-diaminobutane was positively correlated with NADPH.The cloned ppnK and pntAB genes were ligated into pETM6 plasmid and transformed into E. coli BL21(DE3) to form recombinant strains NAP1 and NAP2.Recombinant strains NAP1 and NAP2 could catalyze NADP + to NADPH by expressing pntAB and ppnK.NADPH synthesis and 1,4-diaminobutane yield were shown in Figure 2. The NADPH yield of NAP1 and NAP2 was significantly higher than that of the control strain E. coli BL21(DE3), and similarly, the yield of 1,4-diaminobutane was also increased companied with the elevation of NADPH, showing that improvement of NADPH can enhance the synthesis of 1,4-diaminobutane.The titer of 1,4-diaminobutane in NAP1 and NAP2 was 77 mg/L•DCW and 83 mg/L•DCW, respectively, which was about 27% and 37% higher than that in the control strain E. coli BL21(DE3).In order to furtherly increase the synthesis of NADPH, co-expression plasmid of pntAB and ppnK was transformed into E. coli BL21(DE3) to generate recombinant strain NAP3.The NADPH yield of NAP3 strain was higher, and the 1,4-diaminobutane titer was 95 mg/L•DCW, which was 57% greater than the control strain E. coli BL21(DE3) (Figure 2), indicating that the synthesis of 1,4-diaminobutane was positively correlated with NADPH.
Molecules 2024, 29, x FOR PEER REVIEW 5 of 14 strains NAP7 and NAP8.As shown in Figure 3, with the further improvement of NADPH yield, the synthesis levels of 1,4-diaminobutane were furtherly increased.The titer of 1,4diaminobutane of NAP7 and NAP8 was 88 mg/L•DCW and 98 mg/L•DCW, respectively, which were 47% and 63% higher than the control strain E. coli BL21(DE3).It was proved that overexpression of the genes in the PP pathway could effectively improve NADPH level and 1,4-diaminobutane synthesis.

Effect of Exogenous Supplementation of PLP on the Synthesis of 1,4-Diaminobutane in E. coli
Excessive accumulation of PLP in cells can cause certain toxicity and affect cell growth.Therefore, it is first necessary to determine the effect of PLP concentration on the synthesis of 1,4-diaminobutane.PLP of 100, 200, 300, 400 and 500 µmol/L were added to the medium, and the yield of 1,4-diaminobutane in the fermentation supernatant and OD600 was measured after 24 h of fermentation culture, as shown in Figure 4.When PLP is not added, the titer of 1,4-diaminobutane of E. coli BL21(DE3) is 59 mg/L•DCW, while when PLP is added exogenously at 100 µmol/L, the titer of 1,4-diaminobutane of E. coli BL21(DE3) and OD600 were the highest, respectively reaching 66 mg/L•DCW and 4.However, with the increase of PLP concentration, both the production of 1,4-diaminobutane and OD600 decreased.These results indicated that a reasonable increase of PLP synthesis is beneficial to the synthesis of 1,4-diaminobutane, but not too much, otherwise it will affect the growth of strains and the synthesis of 1,4-diaminobutane.Excessive accumulation of PLP in cells can cause certain toxicity and affect cell growth.Therefore, it is first necessary to determine the effect of PLP concentration on the synthesis of 1,4-diaminobutane.PLP of 100, 200, 300, 400 and 500 µmol/L were added to the medium, and the yield of 1,4-diaminobutane in the fermentation supernatant and OD 600 was measured after 24 h of fermentation culture, as shown in Figure 4.When PLP is not added, the titer of 1,4-diaminobutane of E. coli BL21(DE3) is 59 mg/L•DCW, while when PLP is added exogenously at 100 µmol/L, the titer of 1,4-diaminobutane of E. coli BL21(DE3) and OD 600 were the highest, respectively reaching 66 mg/L•DCW and 4.However, with the increase of PLP concentration, both the production of 1,4-diaminobutane and OD 600 decreased.These results indicated that a reasonable increase of PLP synthesis is beneficial to the synthesis of 1,4-diaminobutane, but not too much, otherwise it will affect the growth of strains and the synthesis of 1,4-diaminobutane.

Enhancing the Synthesis of PLP
The genes pdxJ and dxs, involved in the PLP synthesis pathway, and the genes tktA and talB, responsible for the synthesis of PLP precursors E4P, were cloned into the pETM6 plasmid and transformed into E. coli BL21(DE3) to generate recombinant strains NAP9, NAP10, NAP11 and NAP12.These recombinant strains all could increase PLP synthesis due to expressing pdxJ, dxs, tktA and talB genes related to synthesis of PLP (Figure 1).The results are shown in Figure 5. Overexpression of these genes could increase the synthesis of 1,4-diaminobutane.Overexpression of pdxJ and dxs genes in the PLP synthesis pathway resulted in 1,4-diaminobutane accumulation levels of 67 mg/L•DCW and 69 mg/L•DCW, respectively, with an increase of 11% and 15% compared with the control strain E. coli BL21(DE3).Overexpression of the genes tktA and talB resulted in 1,4-diaminobutane accumulation levels of 86 mg/L•DCW and 80 mg/L•DCW, with an increase of 44% and 35%, respectively, compared with the control strain E. coli BL21(DE3).This showed that increasing the PLP significantly increased 1,4-diaminobutane production.
So as to furtherly enhance PLP synthesis, pdxJ, dxs, tktA and talB were cloned into pETM6 to construct the the PLP synthesis module plasmid pETM6-pdxJ-dxs-tktA-talB.This plasmid was transformed into E. coli BL21(DE3) to create the recombinant strain NAP13.The results showed that when enhancing the expression of PLP synthesis module, the accumulation of 1,4-diaminobutane not only did not increase, on the contrary, the yield of 1,4-diaminobutane decreased by approximately 22% compared with the control strain E. coli BL21(DE3) (Figure 5).At the same time, OD600 also decreased.It was possible that after enhancing the expression of PLP synthesis module, PLP concentration was too high, which led to toxicity to the cells and a reduction in 1,4-diaminobutane production.This was in line with the previous findings that an excessively high PLP concentration did not improve 1,4-diaminobutane production, inversely, it reduced its accumulation.

Enhancing the Synthesis of PLP
The genes pdxJ and dxs, involved in the PLP synthesis pathway, and the genes tktA and talB, responsible for the synthesis of PLP precursors E4P, were cloned into the pETM6 plasmid and transformed into E. coli BL21(DE3) to generate recombinant strains NAP9, NAP10, NAP11 and NAP12.These recombinant strains all could increase PLP synthesis due to expressing pdxJ, dxs, tktA and talB genes related to synthesis of PLP (Figure 1).The results are shown in Figure 5. Overexpression of these genes could increase the synthesis of 1,4-diaminobutane.Overexpression of pdxJ and dxs genes in the PLP synthesis pathway resulted in 1,4-diaminobutane accumulation levels of 67 mg/L•DCW and 69 mg/L•DCW, respectively, with an increase of 11% and 15% compared with the control strain E. coli BL21(DE3).Overexpression of the genes tktA and talB resulted in 1,4-diaminobutane accumulation levels of 86 mg/L•DCW and 80 mg/L•DCW, with an increase of 44% and 35%, respectively, compared with the control strain E. coli BL21(DE3).This showed that increasing the PLP significantly increased 1,4-diaminobutane production.
So as to furtherly enhance PLP synthesis, pdxJ, dxs, tktA and talB were cloned into pETM6 to construct the the PLP synthesis module plasmid pETM6-pdxJ-dxs-tktA-talB.This plasmid was transformed into E. coli BL21(DE3) to create the recombinant strain NAP13.The results showed that when enhancing the expression of PLP synthesis module, the accumulation of 1,4-diaminobutane not only did not increase, on the contrary, the yield of 1,4-diaminobutane decreased by approximately 22% compared with the control strain E. coli BL21(DE3) (Figure 5).At the same time, OD 600 also decreased.It was possible that after enhancing the expression of PLP synthesis module, PLP concentration was too high, which led to toxicity to the cells and a reduction in 1,4-diaminobutane production.This was in line with the previous findings that an excessively high PLP concentration did not improve 1,4-diaminobutane production, inversely, it reduced its accumulation.

Overexpression of 1,4-Diaminobutane Synthesis Module
The bifunctional N-acetyltransferase enzyme encoded by argJ in C. glutamicum catalyzes the conversion of N-acetylornithine to ornithine through deacetylation, and simultaneously transfers the acetyl group produced from this reaction to glutamic acid to form N-acetylglutamic acid, thus facilitating the recycling of the acetyl group [34][35][36].Therefore, the key gene speC gene encoding ODC and argJ were cloned into the pRSM3 plasmid to construct a 1,4-diaminobutane synthesis module plasmid pRSM3-speC-argJ.
The strain NAP14 was constructed by co-transforming the 1,4-diaminobutane synthesis module plasmid pRSM3-speC-argJ and the PLP synthesis module plasmid pETM6-pdxJ-dxs-tktA-talB into E. coli BL21(DE3), and the yield of 1,4-diaminobutane was detected.The results showed that NAP14 produced 86 mg/L•DCW of 1,4-diaminobutane, with a 44% increase compared to the control strain E. coli BL21(DE3) (Figure 6).As ODC is a crucial gene in the synthesis of 1,4-diaminobutane, increasing the expression of ODC in the strain allows for the extensive utilization of PLP and decreases PLP's toxicity.
Afterward, the NADPH synthesis module plasmid pCDM4-pntAB-ppnK and pACM4-zwf-gnd-pgl were constructed and transferred into NAP14 to obtain strain NAP15.The results are shown in Figure 6, the 1,4-diaminobutane titer of NAP15 was 91 mg/L•DCW, with an increase of 51% compared with the control strain E. coli BL21(DE3), which proved that with increasing synthesis of PLP and NAPDH, overexpression of the 1,4-diaminobutane synthesis module could lead to an increase in 1,4-diaminobutane production.At the same time, the density of NAP15 strain decreased, speculating that the four plasmids in the same strain increased the metabolic burden of the strain and inhibited the growth of the strains

Overexpression of 1,4-Diaminobutane Synthesis Module
The bifunctional N-acetyltransferase enzyme encoded by argJ in C. glutamicum catalyzes the conversion of N-acetylornithine to ornithine through deacetylation, and simultaneously transfers the acetyl group produced from this reaction to glutamic acid to form N-acetylglutamic acid, thus facilitating the recycling of the acetyl group [34][35][36].Therefore, the key gene speC gene encoding ODC and argJ were cloned into the pRSM3 plasmid to construct a 1,4-diaminobutane synthesis module plasmid pRSM3-speC-argJ.
The strain NAP14 was constructed by co-transforming the 1,4-diaminobutane synthesis module plasmid pRSM3-speC-argJ and the PLP synthesis module plasmid pETM6-pdxJdxs-tktA-talB into E. coli BL21(DE3), and the yield of 1,4-diaminobutane was detected.The results showed that NAP14 produced 86 mg/L•DCW of 1,4-diaminobutane, with a 44% increase compared to the control strain E. coli BL21(DE3) (Figure 6).As ODC is a crucial gene in the synthesis of 1,4-diaminobutane, increasing the expression of ODC in the strain allows for the extensive utilization of PLP and decreases PLP's toxicity.
Afterward, the NADPH synthesis module plasmid pCDM4-pntAB-ppnK and pACM4zwf-gnd-pgl were constructed and transferred into NAP14 to obtain strain NAP15.The results are shown in Figure 6, the 1,4-diaminobutane titer of NAP15 was 91 mg/L•DCW, with an increase of 51% compared with the control strain E. coli BL21(DE3), which proved that with increasing synthesis of PLP and NAPDH, overexpression of the 1,4-diaminobutane synthesis module could lead to an increase in 1,4-diaminobutane production.At the same time, the density of NAP15 strain decreased, speculating that the four plasmids in the same strain increased the metabolic burden of the strain and inhibited the growth of the strains.

Overexpression of Carbon Source Utilization Pathway Genes
Carbon source is one of the factors that must be considered in fermentation production [37][38][39].In this study, glycerol utilization pathway, glucose utilization pathway, and xylose utilization pathway plasmids were constructed, respectively, and transformed into E. coli BL21(DE3) to construct recombinant strains NAP16, NAP17 and NAP18.The strains were cultured in SOB medium containing 1 g/L glycerol, 1 g/L glucose and 1 g/L xylose, respectively.As shown in Figure 7, the 1,4-diaminobutane titer of NAP16, NAP17 and NAP18 were 93 mg/L•DCW, 102 mg/L•DCW and 78 mg/L•DCW with an increase of 30%, 40% and 24% compared with the control strain E. coli BL21(DE3).These results showed that increasing utilization of the carbon source by overexpressing their genes could significantly increase the yield of the target products, and the recombinant strains overexpressing the glucose pathway genes had the most obvious effect.

Effect of Increasing NADPH and PLP Synthesis on 1,4-Diaminobutane Yield When Utilizing Different Carbon Sources Overexpression of Carbon Source Utilization Pathway Genes
Carbon source is one of the factors that must be considered in fermentation production [37][38][39].In this study, glycerol utilization pathway, glucose utilization pathway, and xylose utilization pathway plasmids were constructed, respectively, and transformed into E. coli BL21(DE3) to construct recombinant strains NAP16, NAP17 and NAP18.The strains were cultured in SOB medium containing 1 g/L glycerol, 1 g/L glucose and 1 g/L xylose, respectively.As shown in Figure 7, the 1,4-diaminobutane titer of NAP16, NAP17 and NAP18 were 93 mg/L•DCW, 102 mg/L•DCW and 78 mg/L•DCW with an increase of 30%, 40% and 24% compared with the control strain E. coli BL21(DE3).These results showed that increasing utilization of the carbon source by overexpressing their genes could significantly increase the yield of the target products, and the recombinant strains overexpressing the glucose pathway genes had the most obvious effect.

Overexpression of Carbon Source Utilization Pathway Genes
Carbon source is one of the factors that must be considered in fermentation production [37][38][39].In this study, glycerol utilization pathway, glucose utilization pathway, and xylose utilization pathway plasmids were constructed, respectively, and transformed into E. coli BL21(DE3) to construct recombinant strains NAP16, NAP17 and NAP18.The strains were cultured in SOB medium containing 1 g/L glycerol, 1 g/L glucose and 1 g/L xylose, respectively.As shown in Figure 7, the 1,4-diaminobutane titer of NAP16, NAP17 and NAP18 were 93 mg/L•DCW, 102 mg/L•DCW and 78 mg/L•DCW with an increase of 30%, 40% and 24% compared with the control strain E. coli BL21(DE3).These results showed that increasing utilization of the carbon source by overexpressing their genes could significantly increase the yield of the target products, and the recombinant strains overexpressing the glucose pathway genes had the most obvious effect.

Fermentation Results of Recombinant Strains
The constructed glucose utilization pathway plasmid, NADPH and PLP synthesis plasmid, and ornithine decarboxylase plasmid were co-transformed into E. coli PUT11(DE3) to construct the strain NAP19.E. coli PUT11(DE3) has been knocked out 11 genes which were associated with the pathways for 1,4-diaminobutane degradation and transport and knocked in DE3 sequence from E. coli BL21(DE3) in PuuR operon site.The fermentation results are shown in Figure 8. NAP19 finally achieved a 1,4-diaminobutane titer of 272 mg/L•DCW, there was a 79% increase compared to the control strain E. coli PUT11(DE3).The results showed that in the strain overexpressing the genes of glucose pathway, enhancing the PLP and NADPH synthesis could increase the yield of 1,4-diaminobutane.However, NAP19 exhibited slower growth compared with the control strain, E. coli PUT11(DE3), which was attributed to the metabolic burden imposed by the presence of four free plasmids containing multi-gene expression cassettes, ultimately impacting the strain's growth efficiency.
means and error bars indicated standard deviation (n = 3 independent experiments.The ** represented p < 0.01 compared with their controls).

Fermentation Results of Recombinant Strains
The constructed glucose utilization pathway plasmid, NADPH and PLP synthesis plasmid, and ornithine decarboxylase plasmid were co-transformed into E. coli PUT11(DE3) to construct the strain NAP19.E. coli PUT11(DE3) has been knocked out 11 genes which were associated with the pathways for 1,4-diaminobutane degradation and transport and knocked in DE3 sequence from E. coli BL21(DE3) in PuuR operon site.The fermentation results are shown in Figure 8. NAP19 finally achieved a 1,4-diaminobutane titer of 272 mg/L•DCW, there was a 79% increase compared to the control strain E. coli PUT11(DE3).The results showed that in the strain overexpressing the genes of glucose pathway, enhancing the PLP and NADPH synthesis could increase the yield of 1,4-diaminobutane.However, NAP19 exhibited slower growth compared with the control strain, E. coli PUT11(DE3), which was attributed to the metabolic burden imposed by the presence of four free plasmids containing multi-gene expression cassettes, ultimately impacting the strain's growth efficiency.

Discussion
NADPH and PLP are the crucial cofactors in the biosynthetic pathway of 1,4-diaminobutane.The construction process of the reported high-yield 1,4-diaminobutane recombinant strains lacked researches on the synthesis of NADPH and PLP.Furthermore, in this study, the effect on the yield of 1,4-diaminobutane was determined by optimizing the PLP and NADPH synthesis pathways in E. coli.
Synthesizing 1 mol 1,4-diaminobutane in the pathway requires the consumption of 2 mol NADPH.Overexpressing key genes involved in NADPH synthesis, such as pntAB and ppnK, along with genes in the PP pathway, could enhance NADPH production and increased the yield of 1,4-diaminobutane.Ultimately, the accumulated amounts in the recombinant strains NAP3 and NAP8 were 95 mg/L•DCW and 98 mg/L•DCW, respectively, showing increases of 57% and 63% compared to the control strains E. coli BL21(DE3).This demonstrated that boosting NADPH synthesis in E. coli could effectively enhance the production of 1,4-diaminobutane.
PLP is the cofactor of ODC.Within a certain range of PLP concentrations, the synthesis of 1,4-diaminobutane is positively correlated with the PLP content in the cells.When

Discussion
NADPH and PLP are the crucial cofactors in the biosynthetic pathway of 1,4-diaminobutane.The construction process of the reported high-yield 1,4-diaminobutane recombinant strains lacked researches on the synthesis of NADPH and PLP.Furthermore, in this study, the effect on the yield of 1,4-diaminobutane was determined by optimizing the PLP and NADPH synthesis pathways in E. coli.
Synthesizing 1 mol 1,4-diaminobutane in the pathway requires the consumption of 2 mol NADPH.Overexpressing key genes involved in NADPH synthesis, such as pntAB and ppnK, along with genes in the PP pathway, could enhance NADPH production and increased the yield of 1,4-diaminobutane.Ultimately, the accumulated amounts in the recombinant strains NAP3 and NAP8 were 95 mg/L•DCW and 98 mg/L•DCW, respectively, showing increases of 57% and 63% compared to the control strains E. coli BL21(DE3).This demonstrated that boosting NADPH synthesis in E. coli could effectively enhance the production of 1,4-diaminobutane.
PLP is the cofactor of ODC.Within a certain range of PLP concentrations, the synthesis of 1,4-diaminobutane is positively correlated with the PLP content in the cells.When the PLP content exceeds a certain range, cell growth is inhibited, leading to a decrease in 1,4-diaminobutane synthesis.Hence, optimizing the PLP content is essential when constructing high-yield 1,4-diaminobutane strains.Overexpressing genes involved in PLP synthesis, such as pdxJ and dxs, and genes involved in the synthesis of PLP precursors E4P, like tktA and talB, could increase the synthesis of 1,4-diaminobutane.However, when pdxJ, dxs, tktA and talB were co-expressed in the recombinant strain NAP13, the accumulation of 1,4-diaminobutane inversely decreased.At the same time, OD 600 also decreased because enhancing the expression of PLP synthesis led to toxicity to the cells.This showed that PLP could promote the biosynthesis of 1,4-diaminobutane in a certain concentration range, otherwise, the biosynthesis of 1,4-diaminobutane was inhibited.
The fermentation of E. coli using glucose, glycerol and xylose was optimized, and the fermentation results showed that the 1,4-diaminobutane produced by the strain using glucose was the highest.Therefore, NADPH and PLP synthesis pathway plasmids, glucose utilization plasmids, and 1,4-diaminobutane synthesis plasmids were transferred to E. coli PUT11(DE3), which knocked out 11 genes associated with 1,4-diaminobutane degradation and transport pathways and incorporated the T7 RNA polymerase of E. coli BL21(DE3).Finally, the yield of the recombinant strain NAP19 reached 272 mg/L•DCW, which was 79% higher than that of the control strain E. coli PUT11(DE3).This suggested that the production of 1,4-diaminobutane could be increased by enhancing the synthesis of NADPH and PLP in the engineering strain.The effect of NADPH and PLP concentrations on 1,4-diaminobutane yield should also be considered.
This study revealed the importance of NADPH and PLP synthesis for the production of 1,4-diaminobutane.Although the yield of 1,4-diaminobutane in recombinant strains was not high, these results proved that the synthesis of NADPH and PLP in recombinant strains could effectively improve the yield of 1,4-diaminobutane, which provided a theoretical basis for the construction of high-yielding 1,4-diaminobutane recombinant strains.

Materials and Methods
4.1.Strain, Plasmid and Culture Conditions E. coli str.K12 substr.MG1655 was used as the starting strain for genome manipulation.E. coli BL21(DE3) and E. coli PUT11(DE3) were used for the synthesis of 1,4-diaminobutane.The constructed strains and genetic phenotypes are shown in Table 1.During plasmid and strain construction, cells were grown on LB solid plates containing appropriate antibiotics.When needed, antibiotics were added at the following concentrations: 50 mg/mL kanamycin (Kan), 100 mg/mL ampicillin (Amp), 25 mg/mL chloramphenicol (Cl) and 50 mg/mL streptomycin (Str).
The target genes in this study were amplified using matched primers using relevant host genomic DNA as a template.Heterologous genes were amplified using primers from E. coli MG1655, C. glutamicum 13032 and B. subtilis genomes as templates, respectively.All of the primers used in this study were listed in Supplementary Table S1.

Plasmids Construction
The pETM6 plasmid and interest genes were digested using SpeI and NdeI and subsequently ligated with a ligase to construct the pETM6 expression plasmid.
The pACM4, pCDM4, pRSM3 and pETM6 plasmids have three major cleavage sites: BlnI, NheI and SalI, and after cleavage they will produce the same CTAG sticky end.Firstly, the interest gene was cloned into the NdeI and SpeI of pETM6.Then, interest gene expression cassette was digested using BlnI and SalI from a constructed plasmid and ligated into between NheI and SalI of the other plasmid by isocaudamers for achieving a tandem linkage of genes (Supplementary Figure S1).The same method was used to tandem genes on pACM4, pCDM4 and pRSM3.The constructed plasmids are shown in Supplementary Table S2.

Shake Flask Culture and Fermentation
The constructed engineering strains were inoculated from solid medium into LB liquid medium containing corresponding antibiotics, and incubated at 37 • C, 220 r/min for 14-16 h as seed culture.The seed was inoculated into 500 mL shake flasks containing fermentation medium according to 1% inoculum volume, and the corresponding antibiotics was added.

Analytical Method
Strain growth was monitored by measuring absorbance at 600 nm (OD 600 ) using a UV spectrophotometer.The dry cell weights (DCW) were determined by heating the samples to a constant weight at 80 • C.
NADPH concentrations were determined based on the glucose dehydrogenase cycle reaction using the CheKine TM Micro Coenzyme II NADP(H) Assay Kit (Abbkine Scientific, CHN.https://www.abbkine.com/product/chekine-micro-coenzyme-%E2%85%A1-nadph-assay-kit-ktb1010/, accessed on 5 June 2024).The strains samples were harvested by centrifugaed and washed with PBS.The pellet cells were added 100 µL of NADPH Extraction Buffer and broken by ultrasonic crushing (power 200 W, ultrasonic 3 s, interval 10 s, repeated 30 times).And then, the extracts were heated at 60 • C for 5 min, 20 µL of Assay Buffer was added, followed by the addition of an equal volume of reverse extraction solution to neutralize it.Vortexed briefly, the samples were centrifuged at 14,000 r/min for 5 min at 4 • C. The supernatant was transferred to a new microcentrifuge tube, and the NADPH content was measured at 450 nm.
1,4-diaminobutane was derived with dansyl chloride so that it can be detected by ultraviolet at 254 nm [40].After 1 mL sample was centrifuged, 500 µL supernatant was taken into a microcentrifuge tube.Added 500 µL saturated NaHCO 3 , and adjusted the pH to 10 with saturated NaOH.Added 1 mL dansyl chloride (5 g/L) for derivative reaction.

Statistical Analysis
The data in this study represented the mean values and standard deviations of three independent experiments.The significant differences in data were determined via a one-way analysis of variance.The value of 0.01 < p < 0.05 indicated a significant difference compared with their controls, and a p value < 0.01 indicated a highly significant difference compared with their controls.

Conclusions
This study optimized the supply of NADPH and PLP in the 1,4-diaminobutane biosynthesis pathway of E. coli and confirmed the positive effects of increased NADPH and PLP synthesis on 1,4-diaminobutane production.Ultimately transferring NADPH and PLP synthetic plasmids and glucose utilization plasmids into the E. coli PUT11(DE3) strain, the resulting strain NAP19 accumulated 272 mg/L•DCW of 1,4-diaminobutane.Increasing the supply of the cofactors PLP and NADPH was shown to be of significant importance for the synthesis of 1,4-diaminobutane.It laid a foundation for the construction of high-yielding 1,4-diaminobutane recombinant strains.

Figure 4 .
Figure 4. Production of 1,4-diaminobutane and OD600 after 24 h fermentation culture with different concentrations of PLP.The concentration of PLP was 100, 200, 300, 400 and 500 µmol/L, and the fermentation supernatant was used to measure the concentration of 1,4-diaminobutane.Data were expressed as means, and error bars indicated standard deviation (n = 3 independent experiments.The * symbol represented 0.01 < p < 0.05, and ** represented p < 0.01 compared with their controls).

Figure 4 .
Figure 4. Production of 1,4-diaminobutane and OD 600 after 24 h fermentation culture with different concentrations of PLP.The concentration of PLP was 100, 200, 300, 400 and 500 µmol/L, and the fermentation supernatant was used to measure the concentration of 1,4-diaminobutane.Data were expressed as means, and error bars indicated standard deviation (n = 3 independent experiments.The * symbol represented 0.01 < p < 0.05, and ** represented p < 0.01 compared with their controls).

Table 1 .
List of strains.