An NADPH-auxotrophic Corynebacterium glutamicum recombinant strain and used it to construct L-leucine high-yielding strain

The NADPH-regeneration enzymes in Corynebacterium glutamicum were inactivated to construct an NADPH-auxotrophic C. glutamicum strain by gene knockout and gene replacement. The resultant NADPH-auxotrophic C. glutamicum XL-1 ΔZMICg::ISm (i.e., strain Leu-1) grew well in the basic medium only with gluconate as carbon source. Replacement of the native glyceraldehyde 3-phosphate dehydrogenase (NAD-GapDHCg) by NADP-GapDHCa from Clostridium acetobutylicum is an effective strategy for producing L-leucine in NADPH-prototrophic strain XL-1 and NADPH-auxotrophic strain Leu-1, whereas the L-leucine yield did not differ significantly between these strains (14.1 ± 1.8 g/L vs 16.2 ± 1.1 g/L). Enhancing the carbon flux in biosynthetic pathway by recombinant expression plasmid pEC-ABNCE promoted L-leucine production, but the shortage NADPH supply limited the L-leucine yield. The mutated promoters of zwf and icdCg were introduced into C. glutamicum with NADP-GapDHCa and pEC-ABNCE increased L-leucine yield (54.3 ± 2.9 g/L) and improved cell growth (OD562 = 83.4 ± 7.5) in fed-batch fermentation because the resultant strain C. glutamicum XL-1 ΔMICg::ISm GCg::GCaPzwf-D1 Picd-D2/pEC-ABNCE (i.e., strain Leu-9) exhibited the proper intracellular NADPH and NADH level. This is the first report of constructing an L-leucine high-yielding strain that reasonably supplies NADPH by optimizing the biosynthetic pathway of NADPH from an NADPH-auxotrophic strain.


Introduction
L-leucine is one of the essential amino acids for animal and human that needs to add into feed or food to meet the requirement of animal and human (Wang et al. 2019a, b). Given the important roles of L-leucine, it has been widely used as feed additive or nutritional supplement in feed or food industry (Luo et al. 2021;Wang et al. 2021). In addition, it is also widely used in chemical and pharmaceutical industry as cosmetic ingredient and medical intermediate, respectively (Ma et al. 2021;Wang et al. 2020). Although L-leucine would be produced by albuminolysis and enzymatic synthesis, microbial fermentation using Corynebacterium glutamicum as work-horse has become the mainstream method to produce L-leucine in industry at present because of the lowest environmental pollution and the best economic effect (Wang et al. 2020). Thus, the L-leucine-producing strain with excellent fermentation performance is vital to increase the final titer and to reduce the production cost. To construct an L-leucine high-yielding strain, however, it not only needs to enhance the carbon flux in L-leucine biosynthetic pathway, but also needs to balance the supply of REDOX cofactor [i.e., NAD(H/ + ) and NADP(H/ + )].
The effect of NADPH in amino acids production has been discussed by Xu et al. (Xu et al. 2019), and Wang et al. pointed out that NADPH shortage is a rate-limiting factor for increasing L-leucine production (Wang et al. 2019a, b). As can be seen from Figs. 1 and 2, molecules NADPH involve in L-leucine biosynthesis at acetohydroxyacid isomeroreductase (AHAIR)-catalyzed reaction and branched-chain amino acid transaminase (BCAT)-catalyzed reaction. In C. glutamicum, four enzymes were reported to involve in NADPH biosynthesis, i.e., glucose-6-phosphate dehydrogenase (Zwf), 6-phosphogluconate dehydrogenase (Gnd), NADP-dependent isocitrate dehydrogenase (NADP-Icd), and malic enzyme (MalE) (Fig. 1) (Xu et al. 2019;Hoffmann et al. 2021). However, the Zwf and Gnd in pentose phosphate (PP) pathway are the main enzymes for NADPH regeneration (Lindner et al. 2018). Increasing the activity of these key enzymes involved in NADPH biosynthesis is one of the most common strategies in constructing NADPH-dependent products high-producing strain . This is mostly achieved by increasing gene copies or replacing the native promoter by a strong promoter, or relieving the feedback regulation of enzyme Li et al. 2016). Although these strategies have acquired certain positive results, the negative factor as the shadow follows the form. For example, enhancement the carbon flux in PP pathway by overexpression of Zwf-and Gnd-coding gene not only improves NADPH supply but also increases the release of CO 2 (Yuan et al. 2021;Jiang et al. 2013). Moreover, irrational supply of NADPH results in the excesses of intracellular NADPH, thus leading to more reactive oxygen species (ROS) production and to injure cells (Xu et al. 2019;Zhu et al. 2021). Therefore, how to optimize the NADPH-supplying pathway under no background noise is important for constructing NADPH-dependent products producing strains with excellent fermentation performance.
In 2018, the researchers in Arren Bar-Even's Lab from Max Planck Institute of Molecular Plant Physiology (Potsdam-Golm, Germany) constructed an Escherichia coli NADPH-auxotroph strain via inactivation of enzymes in NADPH biosynthetic pathway (Lindner et al. 2018), and used it as chassis strain to select formate dehydrogenase variants with high efficiency and specificity toward NADP + (Calzadiaz-Ramirez et al. 2020). They found that the NADPH-auxotroph strain is an effective chassis cells for quantitatively assessing different NADPH-regenerating enzymes and for supporting optimal NADPH production rate by regulating expression levels of NADPH-regenerating enzymes and concentrations of reduced substrates. Referring to the above experimental considerations, therefore, the Fig. 1 Schematic representation of the NADPH auxotrophic C. glutamicum and modified strategies. NADPH and NADH metabolic pathway are shown in blue arrows and brown arrows, respectively. The red arrows represent the extrinsic routes. The symbols "red cross mark" and "green leaf" represent the deletion and the replacement, respectively. G6P, glucose-6-phosphate; 6PGL, 6-phosphogluconolactone; 6PG, 6-phosphogluconate; Ru5P, ribulose-5-phosphate; F6P, fructose-6-phosphate; GAP, glyceraldehyde-3-phosphate; objective of this study was to try to construct an L-leucine high-producing strain based on optimizing NADPH-supplying pathway from C. glutamicum NADPH-auxotroph strain. The main methods of this study involved in three aspects: (1) constructing a C. glutamicum NADPH-auxotroph strain via inactivation of enzymes in NADPH biosynthetic pathway from an L-leucine producing strain C. glutamicum XL-1 (Wang et al. 2019a, b); (2) replacing the native NADdependent glyceraldehyde 3-phosphate dehydrogenase (NAD-GapDH Cg ) by NADP-GapDH Ca from Clostridium Fig. 2 Growth phenotypes of recombinant and original strains under the different carbon sources. Figures from "a" to "h" represent strain XL-1, XL-1 ΔZ, XL-1 ΔM, XL-1 ΔI Cg ::I Sm , XL-1 ΔI Cg , XL-1 ΔZ ΔM, XL-1ΔZ ΔI Cg ::I Sm and Leu-1, respectively. The data represent mean values and standard deviations obtained from three independent cultivations acetobutylicum; and (3) rationally regulating the expression levels of Zwf-coding gene zwf via promoter engineering. As a result, a high-yielding strain C. glutamicum Leu-9 with constructed weak promoter of zwf was obtained, which produced 54.3 ± 2.9 g/L of L-leucine in fed-batch fermentation. Here, we report an effective strategy to construct an L-leucine high-producing strain from an NADPH-auxotrophic strain by optimizing the biosynthetic pathway of NADPH for the first time. The design-based strategy for constructing L-leucine high-yielding strain reported here could serve as a general concept for breeding NADPH-dependent products high-yielding strains.

Strains, growth medium, and culture conditions
Strains used in this study are listed in Table 1. The parental strain C. glutamicum XL-1 was an L-leucine-producing strain constructed by Wang et al. (Wang et al. 2019a, b). E. coli and C. glutamicum were cultivated in Luria-Bertani (LB) medium at 37 ℃ and in LB-glucose (LBG) medium at 30 ℃, respectively (Xu et al. 2016). CgXII medium, consisting of (per liter) 42 g of 3-morpholinopropanesulfonic acid, 20 g of (NH 4 ) 2 SO 4 , 5 g of urea, 1 g of KH 2 PO 4 , 1 g of K 2 HPO 4 ·3H 2 O, 0.25 g of MgSO 4 ·7H 2 O, 0.01 g of CaCl 2 , 0.01 g of FeSO 4 ·7H 2 O, 0.01 g of MnSO 4 ·H 2 O, 0.01 g of ZnSO 4 ·7H 2 O, 0.0002 g of NiCl·6H 2 O, 0.0002 g of biotin, and 0.00003 g of protocatechuic acid, was used for growth experiments (Keilhauer et al. 1993). Thirty-six grams of glucose or the equivalent other carbon sources (calculating based on the energy of combustion among these carbon sources (Lindner et al. 2018)) were used as the carbon source. Appropriately, 50 μg/mL or 25 μg/mL kanamycin (Kan) solution was used to screen the target plasmids and strains. In addition, 1 mmol/L isopropyl β-Dthiogalactopyranoside (IPTG) solution was used to induce gene overexpression.

Construction of C. glutamicum recombinant strains
Plasmids and oligonucleotides used in this study are listed in Table 1 and Supplementary material (Table S1), respectively. Plasmid pEC-XK99E was used for gene expression, and plasmid pK18mobsacB was used for gene deletions and gene replacements in C. glutamicum. The detail methods used for gene modification (i.e., gene expression, gene deletion, and gene replacement) were referred to the published method . To construct Zwf-and/ or MalE-deficient strain, the promoters of gene zwf and/or malE were deleted. The cassettes of gapC Ca with the P gapA promoter, rrnBT1T2 terminator and EcoRI endonuclease was optimized for expression in C. glutamicum and then synthetized by GENEWIZ (Suzhou), Inc. (Suzhou, China). The detail processes of plasmid construction were listed in Supplementary material ( Figure S1)_ENREF_23. For modifying promoter, fragments (~ 400 bp) carrying promoters of the genes zwf, gnd, malE, gapA, and icd were amplified from the chromosome of C. glutamicum XL-1 by PCR with corresponding primers (Table S1). The PCR products after gel purification were digested by the corresponding restriction endonucleases and cloned in the plasmid pDXW-11 (Xu et al. 2011). The resultant plasmids were transferred into C. glutamicum by electroporation, and the target recombinants were selected referring to the published method (van der Rest et al. 1999).

Preparation of crude enzyme and enzyme activity assays
The cells in the logarithmic growth were collected after centrifugation for 10 min at 5000 × g, and then were disrupted by sonication. Following, fragmentized liquid was centrifuged by centrifuge at 4 °C and 10,000 × g for 30 min, and then the cell-free supernatants were used as crude enzyme to determine the enzyme activities. Protein concentrations were analyzed using the Bradford Protein Quantification Kit (Vazyme, Nanjing, China) with bovine serum albumin as standard. The enzyme activity assays were done in

Quantification of intracellular pyridine nucleotides
The intracellular pyridine nucleotides (i.e., NADH/NAD and NADPH/NADP) of cells cultivated for 36 h were extracted referring to the published method (Faijes et al. 2007), and the concentration was detected using NAD/NADH Quantification Colorimeteric Kit or NADP/NADPH Quantification C. glutamicum α-AB r 2-TA r SG r β-HL r Ile -Met -, a L-leucine-producing strain derived from strain ATCC13032 (Wang et al. 2019a, b) XL-1 Strain XQ-9 derivative with in-frame deletion of ltbR (Wang et al. 2020) Leu-1 An NADPH-auxotrophic strain derived from strain XL-1 with inactivated Zwf and MalE as well as replacement with NAD-dependent Icd This study Leu-2 The native NAD-dependent GapDH was replaced by NADP-dependent GapDH in strain Leu-1 chromosome This study Leu-3 The native promoter of gene zwf was replaced by promoter P zwf -D5 in strain Leu-2 chromosome and harboring plasmid pEC-ABNCE

This study
Leu-4 The native promoter of gene zwf was replaced by promoter P zwf -D1 in strain Leu-2 chromosome and harboring plasmid pEC-ABNCE This study  The native promoter of gene zwf was replaced by promoter P zwf -S6 in strain Leu-2 chromosome and harboring plasmid pEC-ABNCE This study Leu-6 The native promoter of gene icd was replaced by promoter P icd -D2 in strain Leu-2 chromosome and harboring plasmid pEC-ABNCE This study  The native promoter of gene icd was replaced by promoter P icd -S7 in strain Leu-2 chromosome and harboring plasmid pEC-ABNCE

This study
Leu-8 The native promoter of gene icd was replaced by promoter P icd -T3 in strain Leu-2 chromosome and harboring plasmid pEC-ABNCE This study Leu-9 The native promoters of genes zwf and icd were replaced by promoter P zwf -D1 and P icd -D2 in strain Leu-2 chromosome and harboring plasmid pEC-ABNCE

This study
Leu-10 The native promoters of genes zwf and icd were replaced by promoter P zwf -D1 and P icd -S7 in strain Leu-2 chromosome and harboring plasmid pEC-ABNCE

This study
Leu-11 The native promoters of genes zwf and icd were replaced by promoter P zwf -D1 and P icd -T3 in strain Leu-2 chromosome and harboring plasmid pEC-ABNCE This study pK18mobsacB/∆P icd ::P icd-M Integration vector for replacement of the promoter of icd gene by the mutated promoter of icd gene (i.e., P icd -D2, P icd -S7, and P icd -T3) This study Colorimeteric Kit (BioVision, Inc., Milpitas, CA) based on the manufacturer's instructions, respectively.

Analytical methods
A sample was taken from the shake flasks at the right time.
The sample was used to analyze cell growth using a spectrophotometer at 562 nm after an appropriate dilution. In addition, glucose concentration was determined using an SBA-40E immobilized enzyme biosensor (Shandong, China). The concentration of L-leucine was determined by high-performance liquid chromatography (HPLC) using Agilent 1200 system (Agilent Technologies, Santa Clara, CA, USA) according to the procedure described by Wang et al. (Wang et al. 2020).

Construction and characterization of an NADPH-auxotrophic C. glutamicum strain
In C. glutamicum, there are four enzymes that are reported to involve in NADPH regeneration ( Fig. 1) (Xu et al. 2019;Hoffmann et al. 2021). In order to construct an NADPHauxotrophic C. glutamicum strain, we aimed to inactivate the Zwf and the MalE as well as to replace the native NADP-Icd Cg by NAD-Icd Ca from S. mutans. In this study, the Gnd was not inactivated because the activity of Gnd was controlled by the availability of 6-phosphogluconate, which was the product in Zwf-catalyzed reaction (Fig. 1).
In addition, the strain with the activated Gnd can maintain the cell growth during using gluconate as carbon source because gluconate can convert to 6-phosphogluconate without Zwf participation (Lindner et al. 2018). The cell growth of Zwf-deficient strain (i.e., C. glutamicum XL-1 ΔZ) was significantly decreased during growth on most carbon source except on gluconate, whereas the cell growth of MalE-deficient strain (i.e., C. glutamicum XL-1 ΔM) had little change during growth on the test carbon sources except on succinate (Fig. 2a-c). It is worth noting that replacement of the native NADP-Icd Cg by NAD-Icd Sm hardly affected growth on all carbon sources (Fig. 2d), but disruption of NADP-Icd Cg severely inhibited growth on most other carbon sources except on succinate (Fig. 2e). Next, we integrated the above gene modifications with the disruption of MalE and/or replacement with NAD-Icd Sm . As can be seen from Fig. 2b, f, and g, further disruption of MalE in strain XL-1 ΔZ was not significantly impaired cell growth on any of the carbon sources, but further replacement with NAD-Icd Sm in strain XL-1 ΔZ almost abolished cell growth on most carbon source except on gluconate. Deletion of zwf and malE as well as replacement with icd Sm (i.e., C. glutamicum XL-1 ΔZMI Cg ::I Sm , also known as strain Leu-1) completely abolished cell growth on most carbon source except on gluconate (Fig. 2h). Different from the cell growth, the L-leucine yield of these recombinant strains was obviously decreased with modification of the enzyme involved in NADPH regeneration except the strain C. glutamicum XL-1 ΔM during cultivated in fermentation medium (Table 2). Thus, we selected strain Leu-1 as NADPH-auxotrophic C. glutamicum strain to optimize NADPH-supplying pathway for constructing L-leucine high-producing strain.

Replacement of glyceraldehyde 3-phosphate dehydrogenase to restore NADPH regeneration and L-leucine production
As mentioned above, the strain Leu-1 is an NADPH-auxotrophic C. glutamicum strain, thus abolishing cell growth and L-leucine production on glucose (Fig. 2). In 2018, NADPHauxotrophic E. coli strain was used as chassis cells to assess the capacity of different enzymes to biosynthesize NADPH (Lindner et al. 2018). In this study, we tried to use strain Leu-1 as chassis cells to investigate the effect of exogenous NADPH-supplying pathway on L-leucine production. Previous researchers indicated that replacing the native NADdependent glyceraldehyde-3-phosphate dehydrogenase (NAD-GapDH) with the nonphosphorylating NADP-GapDH allows cells to produce 2 molecules of NADPH rather than NADH per molecule of glucose (Martinez et al. 2008). Thus, we replaced the native NAD-GapDH with the NADP-GapDH from C. acetobutylicum in strain Leu-1, and the resultant strain C. glutamicum XL-1 ΔZMI Cg ::I Sm G Cg ::G Ca (i.e., strain Leu-2) restored the regenerative power of NADPH using glucose as carbon source ( Table 2). As a control, we also replaced the NAD-GapDH Cg with NADP-GapDH Ca in strain XL-1, and the resultant strain C. glutamicum XL-1 G Cg ::G Ca obviously increased the intracellular NADPH level (Table 2). In addition, the maximum specific growth rate (μ max ) for the parental strain XL-1 and the GapDH-modified strains Leu-2 and XL-1 G Cg ::G Ca were 0.35 h -1 , 0.32 h -1 , and 0.19 h -1 , respectively. And the final OD 562 of these above strains are listed in Table 2. As expected, the strain Leu-2 produced 16.2 ± 1.1 g/L of L-leucine because of the restoration of NADPH supply, which was slightly higher than that of parental strain XL-1 (15.3 ± 1.2 g/L of L-leucine) (Fig. 3). It is worth noting that the productivity (i.e., the L-leucine yield per OD 562 ) of strain Leu-2 was obvious higher than that of strain XL-1 (0.468 vs 0.387 g/(L•OD 562 ); Fig. 3). In addition, strain XL-1 G Cg ::G Ca only produced 14.1 ± 1.8 g/L of L-leucine (Fig. 3), even though the intracellular NADPH level in strain XL-1 G Cg ::G Ca was higher than that in strain Leu-2 and strain XL-1 (Table 2). However, the productivity of strain XL-1 G Cg ::G Ca was 0.534 g/(L•OD 562 ), which was 14.1% and 40.0% higher than that of strain Leu-2 and strain XL-1, respectively (Fig. 3).

Enhancing the carbon flux in L-leucine biosynthetic pathway to further promoting L-leucine production
There are seven enzyme-catalyzed reactions from pyruvate in L-leucine biosynthetic pathway (Fig. 1). In order to enhance the carbon flux in L-leucine biosynthetic pathway, we tried to over-express the genes ilvBN (encoding acetohydroxyacid synthase, AHAS, represented by "BN"), ilvC (encoding AHAIR, represented by "C"), leuA (encoding α-isopropylmalate synthase, IPMS, represented by "A"), and ilvE (encoding BCAT, represented by "E") by expression vector pEC-XK99E. In addition, the mutated AHAIR (encoded by ilvC M , represented by "C M "), which uses NADH as factor rather than NADPH, was also over-expressed. The resulted plasmids pEC-ABNCE and pEC-ABNC M E were transferred into strain Leu-2, and the cell growth, L-leucine yield, as well as the intracellular NADH and NADPH level in the resultant recombinant strains were determined. The resultant strain Leu-2/pEC-ABNCE showed the similar cell growth with the parental strain XL-1, whereas the resultant strain Leu-2/pEC-ABNC M E obviously decreased the cell growth as compared with the parental strain XL-1 (Table 2). In addition, the intracellular NADPH level of strain Leu-2/pEC-ABNCE was decrease to 2.56 × 10 −4 nmol/ (10 4 cell), which was 64.3% lower than that of strain Leu-2 (Table 2). However, the strain Leu-2/pEC-ABNC M E exhibited a slight decrease in the intracellular NADPH level as compared with the strain Leu-2 ( Table 2). As expected, strain Leu-2/pEC-ABNCE produced 26.3 ± 1.7 g/L of L-leucine in 72-h shake flask fermentation, which was about 1.6 and 1.2 times higher than that of strain Leu-2 (16.2 ± 1.1 g/L) and strain Leu-2/pEC-ABNC M E (22.6 ± 2.3 g/L), respectively (Fig. 3). It is worth noting that strain Leu-2/pEC-ABNC M E showed the highest productivity of L-leucine [0.743 g/(L•OD 562 )], followed by the strain Leu-2/pEC-ABNCE [0.646 g/(L•OD 562 )], and strain Leu-2 [0.468 g/(L•OD 562 )].

Analyzing the promoter activity of key enzymes involved in NADPH regeneration to determine the key pathway for NADPH regeneration
As mentioned above (Table 2, Fig. 3), strain Leu-2/pEC-ABNC M E showed the high intracellular NADPH level and L-leucine productivity despite of the low L-leucine yield.
In contrast, strain Leu-2/pEC-ABNCE exhibited the low intracellular NADPH level and L-leucine productivity as well as the high L-leucine yield. We speculated that the L-leucine production in strain Leu-2/pEC-ABNCE was limited because of the shortage of NADPH. Thus, future research should focus on improving NADPH supply by optimizing the NADPH biosynthetic pathway. To determine the promoter activity of zwf, gnd, icd, and malE in C. glutamicum XL-1, their upstream regions of these genes were cloned in the promoter-probe vector pDXW-11, giving rise to the constructs pDXW-11-P zwf , pDXW-11-P gnd , pDXW-11-P icd , and pDXW-11-P malE (Fig. 4a). Based on the analysis results from BDGP (Berkeley Drosophila Genome Project, https:// www. fruit fly. org/ seq_ tools/ promo ter. html), BPROM (http:// linux1. softb erry. com/ berry. phtml? topic= bprom & group= progr ams& subgr oup= gfindb) and reported by Pátek and Nešvera (Miroslav Patek & Nesvera 2013), about 400 bp fragment upstream from initiation codon of these genes included all of promoter elements, and the potential -10 and -35 regions as well as the transcriptional start site are summarized in Table 3. The transcriptional activity of these cloned fragments including promoters in C. glutamicum was investigated using the cat reporter gene (encoding CAT) in the extracts of cells cultivated in LBG medium. The CAT-coding gene in plasmid pDXW-11 was successfully expressed under the control of  Fig. 4 The promoter activity of key enzymes involved in NADPH regeneration. (a) The strategies for constructing the promoter-probe vectors. (b) Analyzing the CAT expression of strains under the different promoters by SDS-PAGE. Lane 1 E. coli pDXW-11; Lane 2 E. coli pDXW-11-P gapA ; Lane 3 E. coli pDXW-11-P malE ; Lane 4 E. coli pDXW-11-P icd ; Lane 5 E. coli pDXW-11-P zwf ; Lane 6 E. coli pDXW-11-P gnd ; Lane M Protein marker. The blot in the red box is the CAT these mentioned promoters (Fig. 4b). As can be seen from Table 3, in addition, the promoters P zwf and P gnd were strong [0.365 ± 0.038 U/(mg protein) and 0.406 ± 0.025 U/(mg protein), respectively], whereas the promoter P malE was rather weak [0.010 ± 0.003 U/(mg protein)] and the promoter P icd was of intermediate strength [0.092 ± 0.012 U/(mg protein)]. These results indicated that the PP pathway is the key pathway for NADPH regeneration and the tricarboxylic acid (TCA) cycle comes second, whereas the transhydrogenase-like cycle hardly works in strain XL-1.

Constructing the mutant promoters of the zwf and/ or icd Cg genes to balance the intracellular NADPH and NADH level
As mentioned above (Fig. 3), although the strain Leu-2/ pEC-ABNC M E showed the lower L-leucine yield than that of strain Leu-2/pEC-ABNCE, it showed the higher productivity than that of strain Leu-2/pEC-ABNCE. These results implied that the strain Leu-2/pEC-ABNC M E accumulates excess intracellular NADPH level but the strain Leu-2/pEC-ABNCE lacks adequate NADPH for L-leucine production. It should be noted that the promoter of gapA gene (i.e., P gapA ) is a strong promoter because the strain with pDXW-11-P gapA showed the high CAT activity [0.753 ± 0.023 U/(mg protein)] (Table 3). In order to efficiently supply NADPH for L-leucine production, the promoters of the zwf and/or icd Cg genes (i.e., P zwf and P icd ) were mutated to regulate the expression level of genes controlled by these promoters. In this study, the − 10 region of P zwf and P icd was selected as modified object to regulate promoter activity, and the promoter activity was analyzed the CAT activity using the promoter-probe vector pDXW-11. As can be seen from Table 3 and Figure S2, the mutated promoters with base substitution in conservative positions of -10 regions showed the low activity, whereas base substitution in nonconservative positions showed the positive effects on increasing promoter activity. For optimizing promoter of gene zwf, the mutated promoters P zwf -D5, P zwf -D1, and P zwf -S6 (with about 50%, 30%, and 10% of activity of native-type P zwf , respectively) were selected for further investigating. In addition, the mutated promoters P icd -T3, P icd -S7, and P icd -D2 (with about 2.0-, 3.0-and 5.0-fold stronger than native-type P icd , respectively) were selected for optimizing promoter of gene icd.
Following, the mutated P zwf were introduced into strain Leu-2 to control the expression of zwf gene, and the mutated P icd replaced the native promoter of icd Cg gene in strain Leu-2 to control the expression of icd Sm gene. Subsequently, the overexpression plasmid pEC-ABNCE was transferred into these recombinant strains. The resultant strains with the mutated P zwf [i.e., C. glutamicum Leu-2 P zwf -D5/pEC-ABNCE (strain Leu-3), C. glutamicum Leu-2 P zwf -D1/pEC-ABNCE (strain Leu-4), and C. glutamicum Leu-2 P zwf -S6/pEC-ABNCE (strain Leu-5)] showed the increased yield and productive of L-leucine as compared with the strain Leu-2 (Fig. 5a). Among them, the strain Leu-4 showed the best fermentation performance, which produced 28.6 ± 2.4 g/L of L-leucine with a productivity of 0.769 g/(L•OD 562 ) in 72-h shake flask fermentation. In addition, the strains with the mutated P icd [i.e., Table 3 Activity of promoters assayed by measuring the specific activity of chloramphenicol acetyltransferase (CAT) in cell extracts of C. glutamicum a The bold and italic letters represent the -35 region, the letters with underline represent -10 region, and the bold letters with underline represent the transcriptional start site b Nucleotide substitutions are capital letters and in bold c It represents the distance between the transcriptional start site and the initiation codon d The key elements of promoter were deduced from programs BDGP and BPROM e The key elements of promoter were referred to the previous results reported by Pátek -tatAAt 0.462 ± 0.027 -C. glutamicum Leu-2 P icd -T3/pEC-ABNCE (strain Leu-6), C. glutamicum Leu-2 P icd -S7/pEC-ABNCE (strain Leu-7), and C. glutamicum Leu-2 P icd -D2/pEC-ABNCE (strain Leu-8)] exhibited a 5-20% decrease in L-leucine production and a 10-30% increase in cell growth (Table 2). However, replacement of the native P icd by the P icd -T3 in the strain Leu-4 showed the best L-leucine yield and cell growth (Fig. 5a). The resultant strain C. glutamicum Leu-2 P zwf -D1 P icd -T3/pEC-ABNCE (i.e., strain Leu-9) produced 32.8 ± 1.7 g/L of L-leucine, and its OD 562 reached to 41.3 ± 4.2 during cultivated in shake flask for 72 h. In addition, the strain Leu-9 has been a slight increase in the intracellular NADH level and the ratio of NADH/NAD + (Table 2). However, the introduction of the strong promoters P icd -S7 and P icd -D2 to control icd Sm gene expression (i.e., strain Leu-10 and Leu-11) was harmful to L-leucine production despite increasing the cell growth and the intracellular NADH level (Fig. 5b, Table 2). It should be noted that the strain Leu-9 also showed the good fermentation performance in fed-batch fermentation, in which L-leucine began to be secreted into the broth at the exponential phase and continuously increased to a final yield of 54.3 ± 2.9 g/L after 72 h (Fig. 6). Based on these results, we speculated that the strain Leu-9 with P zwf -D1 and P icd -T3 to respectively control the expression of zwf and icd Sm exhibited the proper intracellular NADPH and NADH level, and thus beneficial to L-leucine production.

Discussion
For the first time, we report an NADPH-auxotrophic C. glutamicum recombinant strain and use it to construct L-leucine high-yielding strain. To do this, an L-leucineproducing strain C. glutamicum XL-1 was consecutively modified to give strain XL-1 with high-efficiency of L-leucine production, for example, deletion of the native promoter of the NADPH-regenerating enzymes-coding genes to construct a C. glutamicum NADPH-auxotroph strain, introduction of the nonphosphorylating NADP-GapDH to restore NADPH regeneration, and optimization of the promoters of the zwf and/or icd Cg genes to balance the intracellular NADPH and NADH level. As a result, a L-leucine high-producing strain C. glutamicum Leu-9 was obtained, which produced 32.8 ± 1.7 g/L of L-leucine in 72-h shake flask fermentation and produced 54.3 ± 2.9 g/L of L-leucine with a productivity of 0.651 ± 0.063 g/ (L•OD 562 ) in fed-batch fermentation. These results are encouraging, as far as we know (Table 4), indicating that strain Leu-9 is a competitive platform strain for L-leucine production. These results also inferred that optimization of the biosynthetic pathway of NADPH from an NADPHauxotrophic strain is easy to reasonably supply NADPH, thus increasing the production of NADPH-dependent valuable chemicals.
In C. glutamicum, there are four enzymes involved in NADPH biosynthesis, i.e., Zwf, Gnd, NADP-Icd, and MalE ( Fig. 1) (Xu et al. 2019;Hoffmann et al. 2021). It is clear from this study of the roles of NADPH-regenerating enzymes that different NADPH-regenerating enzymes show a huge difference in catalyzing the NADPH regeneration in C. glutamicum (Table 2 and Fig. 2). As in previous reports (Lindner et al. 2018), our results show that Zwf and Gnd in PP pathway are the main enzymes for NADPH regeneration, in which the promoters P zwf and P gnd from strain XL-1 show the strong activity (Table 3). It is worth noting that the native P icd from strain XL-1 show the weak activity (Table 3), which is different from previous reports (Han et al. 2008;Aich et al. 2001). Han et al. pointed out that the transcript level of icd from C. glutamicum R is high (i.e., 10 ~ 25) in the presence of most of carbon sources, except citrate, succinate, and malate (Han et al. 2008). In addition, Aich et al. agreed that the icd promoter from E. coli is a strong promoter (Aich et al. 2001). Previous studies have already indicated that the activity of icd promoter is regulated by secondary substances and accessory proteins (Shechter et al. 2003;Prost et al. 1999). Moreover, base mutation in promoter including -10 region, -35 region, and upstream of -35 region will lead to change the promoter activity (Holatko et al. 2009;Vasicova et al. 1999). Given that the strain XL-1 is mutant strain engineered by repeated random mutagenesis (Wang et al. 2019a, b), random genetic mutation may be happen in some structure genes and/or icd promoter and furthermore changes the amount of secondary substances and accessory proteins as well as promoter activity. As expected, inactivation of these NADPH-regenerating enzymes in C. glutamicum leads to no NADPH biosynthesis in the resultant strain Leu-1 except using gluconate as carbon source (Table 2 and Fig. 2). The similar results are also found in previous research, in which the NADPHauxotroph strain E. coli NADPHaux grew well only using gluconate as carbon source (Lindner et al. 2018).
Biosynthesis of 1 molecule of L-leucine needs 2 molecules of NADPH (Wang et al. 2019a, b). To meet the requirement of NADPH for L-leucine production, the native NAD-GapDH was replaced by the NADP-GapDH in strain Leu-1 and original strain XL-1. Since NADP-GapDH allows cells to produce 2 molecules of NADPH per molecule of glucose (Martinez et al. 2008), the resultant strain Leu-2 restored the cell growth and L-leucine production using glucose as carbon source (Table 2, Fig. 3). However, the L-leucine productivity of strain XL-1 G Cg ::G Ca was higher than that of strains Leu-2 and XL-1, even though the L-leucine yield of strain XL-1 G Cg ::G Ca was lower than that of strain Leu-2 and strain XL-1 (Table 2, Fig. 3). In addition, the strain Leu-2 showed the lower the cell growth than that of strain XL-1 despite the higher L-leucine production ( Table 2). We speculate that the NADPH supply is inadequate to meet the requirement of NADPH for L-leucine production in strain XL-1, whereas strains Leu-2 and XL-1 G Cg ::G Ca accumulates excess NADPH and thus limits the cell growth. This speculation has Table 4 Overview of the production of L-leucine by metabolic engineered C. glutamicum a Estimated from reference. The relation between DCW and OD562 was referred to the formula: DCW (g/L) = 0.57 × OD 562 + 0.23 reported in previous study (Wang et al. 2020) b No computed data  (Luo et al. 2021) been proved during enhancement of the carbon flux in L-leucine biosynthetic pathway in strain Leu-2. As can be seen from Table 2, strain Leu-2/pEC-ABNCE showed the similar cell growth with the parental strain XL-1 and the higher than that of strain Leu-2. By contrast, the resultant strain Leu-2/ pEC-ABNC M E obviously decreased the cell growth as compared with the parental strain XL-1 ( Table 2). The difference between pEC-ABNCE and pEC-ABNC M E is the AHAIRcoding gene ilvC that needs NADPH as cofactor to catalyze the biosynthesis of α,β-dihydroxyisovalerate. However, the mutated AHAIR (encoded by gene ilvC M , abbreviated to C M ) uses NAD as cofactor to catalyze the biosynthesis of α,βdihydroxyisovalerate because of the mutant AHAIR with three amino acid mutations (i.e., S34G, L48E, and R49F) (Hasegawa et al. 2013). As a result, the intracellular NADPH level of strain Leu-2/pEC-ABNC M E was slightly decreased while that of strain Leu-2/pEC-ABNCE was significantly decreased (Table 2). Although strain Leu-2/pEC-ABNCE produced more L-leucine titer than strain Leu-2/pEC-ABNC M E (26.3 ± 1.7 g/L vs 22.6 ± 2.3 g/L), but the productivity of L-leucine was lower than that of strain Leu-2/pEC-ABNC M E [0.646 g/(L•OD 562 ) vs 0.743 g/(L•OD 562 )]. The results demonstrated that the NADPH supply in strain Leu-2/ pEC-ABNCE is still a limiting factor for L-leucine production. In order to increase NADPH supply in strain Leu-2/ pEC-ABNCE, the key enzymes in NADPH regeneration (i.e., Zwf and Icd) were reasonably modified again. To do this, the − 10 region in promoters of the zwf and/or icd Cg genes (i.e., P zwf and P icd ) was mutated to regulate the expression level of zwf and icd Cg . Consistent with the previous results (Holatko et al. 2009;Vasicova et al. 1999), the mutated promoters with base substitution in conservative positions of -10 regions showed the low activity, whereas base substitution in nonconservative positions showed the positive effects on increasing promoter activity (Table 3 and Figure S2). To optimize the expression level of zwf and icd Cg for balancing the intracellular NADPH and NADH level, three mutated promoters of P zwf (i.e., P zwf -D5, P zwf -D1, and P zwf -S6) and three mutated promoters of P icd (i.e., P icd -T3, P icd -S7, and P icd -D2) were selected for further investigating the effect on L-leucine production. As can be seen from Table 2, the intracellular NADPH level was decreased with the decrease of promoter activity of P zwf , whereas the highest L-leucine yield (i.e., 28.6 ± 2.4 g/L) was found in strain Leu-4 with P zwf -D1. It should be noted that strain Leu-4 showed the lower OD 562 than that of strain Leu-2/pEC-ABNCE and not significantly increased the L-leucine production as compared with strain Leu-2/pEC-ABNCE (by only about 8.7%). These results indicated that excess NADPH supply is bad for L-leucine production. The similar results were also found in previous reports, in which excess NADPH supply will disturb glucose uptake and cell growth and thus inhibits the production of L-lysine or L-threonine (Xu et al. 2019;Liu et al. 2019). It is worth noting that the L-leucine yield was not increased but the cell growth was obviously increased during increasing the promoter activity of P icd ( Table 2). The similar results were also found in the next study that the mutated P icd with different activity was icd that the L-leucine yield was not increased but the cell growth was obviously increased during increasing the promoter activity introduced in the strain Leu-4 (Table 2 and Fig. 5a). One of the reasons is that more NADH was regenerated during the increase of the activity of Icd Sm (Table 2 and Fig. 5b), and thus to meet the demand of cell growth for energy (Yumnam et al. 2021). It should be noted that the strains with high Icd Sm activity showed the low yield and productivity of L-leucine because of more carbon source was used for cell growth.
In conclusion, an NADPH-auxotrophic C. glutamicum recombinant strain can be constructed by the inactivation of Zwf and MalE as well as replacement of icd Cg by icd Sm in C. glutamicum. The NADPH-auxotrophic C. glutamicum strain Leu-1 can be used as chassis cells for constructing L-leucine high-yielding strain based on optimizing the biosynthetic pathway of NADPH. After a series of genetic modifications in strain XL-1, an L-leucine high-yielding strain Leu-9 was obtained, which produced 54.3 ± 2.9 g/L with a productivity of 0.651 ± 0.063 g/(L•OD 562 ) in fed-batch fermentation.
The oligonucleotides used in this study; Strategy used for the construction of the integrative plasmids used for promoter deletion and gene replacement; Effects of mutations in the -10 region of P zwf (a) and P icd (b) on promoter activity.