Metabolic Engineering of Bacillus subtilis for 2.3-BDO production by introducing an exogenous NADPH/NADP+ regeneration system

Background: Generally, glucose is transformed into pyruvate from glycolysis before the target products acetoin and 2,3-butanediol (2,3-BDO) are formed. Pentose Phosphate Pathway (PPP) is an ine�cient synthetic pathway for pyruvate production from glucose in Bacillus subtilis. Previously, it was found that engineered PPP in B. subtilis unbalanced NADH and NADPH regeneration systems and affected acetoin and 2,3 -BDO production. Results: In this study, metabolic engineering strategies were proposed to redistribute carbon �ux to 2,3-BDO via reconstructing intracellular cofactors regeneration systems. Firstly, extra copies of glucose dehydrogenase (GDH)and an exogenous NADPH-dependent 2,3-BDO dehydrogenase (TDH) were introduced into the GRAS strain B. subtilis 168 to introduce an exogenous NADPH/NADP + regeneration system and broaden 2,3-BDO production pathway. It was found that overexpressing the NADPH/NADP + regeneration system effectively improved 2,3-BDO production and inhibited NADH-dependent by-products accumulation. Subsequently, the disruption of lactate dehydrogenase (encoded by ldh ) by insertion of the transcriptional regulator ALsR, essential for the expression of alsSD (encoding two key enzymes for the conversion of pyruvate to acetoin) in B. subtilis, resulted in the recombinant strain in which alsSD was overexpressed and the pathway to lactate was blocked simultaneously. On fermentation by the result engineered strain, the highest 2,3-BDO concentration increased by18.43%, while the titers of main byproducts acetoin and lactate decreased by 22.03% and 64%, respectively. Conclusion: In this study, it shows that engineering PPP and reconstructing intracellular cofactors regeneration system could be an alternative strategy in the metabolic engineering of 2,3-BDO production in B. subtilis .

Background 2,3-butanediol 2.3-BDO is an important platform compound and it has important applications in industry [1][2][3].As a potential environmentally friendly fuel, the heating value of 2.3-BDO is 27.2 kJ/g [4] comparing with ethanol 29.055 kJ/g and methanol 22.081 kJ/g [5], it can mixed with gasoline in any proportion.Due to the shortage of fossil fuels, the production of 2,3-BDO is catching more attentions [1].
In recent years, production of 2, 3-BDO by microorganisms has been greatly developed by regulating NADH regeneration system.Yang et al. improved 2,3-BDO production by 22.7% in B.amyloliquefaciens via rebuilding NADH/NAD + regeneration system by co-overexpression of Glyceraldehyde-3-Phosphate Dehydrogenase /2,3-butanediol Dehydrogenase [14], and increased 2,3-BDO titer by 25.5% in B. subtilis via introducing an extra NADH regeneration system and disrupted NADH oxidase [15].Fu et al. successfully enhanced the 2,3-BDO titer by 13.6% with the introduction of transhydrogenase to improve the NADH yield from NADPH in B. subtilis [16].Dai et al. simultaneously increased 2,3-BDO titer by the higher NADH/NAD + ratio in Paenibacillus polymyxa with additional vitamin C in the fermentation medium [17].These regulatory strategies were all based on regulating NADH levels.In this study, we payed more attentions to regulate a different cofactor NADPH levels and tried to improve 2, 3-BDO titer by reconstructing a NADPH/NADP + regeneration system.
Pyruvates could be produced via two pathways: the Glycolysis Pathway (EMP) and Pentose Phosphate Pathway PPP .In the EMP, one glucose produces two pyruvates accompanying with two molecules of NADH [1].While, through the PPP, pyruvate is produced accompanying with NADPH production [18].In our previous study, it was found that acetoin could be enhanced by engineering the PPP pathway and disturbing the coenzyme balance of NADH/NADPH [19].In this study, we tried Introducing an exogenous NADPH-dependent 2, 3-butanediol dehydrogenase to construct the NADPH/NADP + regeneration system in engineered B. subtilis strains Overexpression of GDH increased the level of NADPH and decreased NADH levels which repressed the conversion of acetoin to 2,3-BDO.To enhance the carbon ux to 2, 3-BDO , we tried to introduce an exogenous NADPH-dependent 2,3-BDO dehydrogenase [21] (coding gene tdh in Clostridium autoethanogenum DSM 10061 into engineered B.subtilis to construct an extra NADPH/NADP + regeneration system with help of GDH, and observed their effects on 2,3-BDO fermentation. In eukaryotes, poly A tails are stretch adenine bases in the 3' end of RNA, and it was found that when the poly(A) tails become short enough, the mRNA could be degraded.It was also reported that the different of poly(A) tails had a great effect on the stability of mRNA of the enzyme, which plays a key role in its overproduction process [25][26][27].
In this study, to improve the e ciency of carbon ow from acetoin to 2,3-BDO, poly (A) tails (AAATTT, TTTAAATTT, AAATTTAAATTT, TTTTTTAAATTT and TTTTTT)were separately added to the 3' end of the tdh to improve its transcription abundance and TDH production in B. subtilis, and the resultant strains named BM6, BM7, BM8, BM9 and BM10, respectively.The results were showed in Figure 2. The transcription abundance of tdh in strains BM6, BM7, BM8 and BM9 were all decreased, compared with strains BM5 without any poly (A/T) tails, and the lower transcription abundance resulted in lower TDH activities in strains BM6, BM7, BM8 and BM9.However, compared with strains BM5, the transcription abundance of TDH in strain BM10 increased by 85 %, which resulted in the TDH activity reached to 180.35 mu/mg and increased by 32.6 %.
Subsequently, the TDHR5 and gdh with different promoters were co-overexpressed in B. subtilis 168 resulting in recombinant strains BM11, BM12, BM13 and BM14 (Tale 3).It was observed that fermentation times were shortened signi cantly, the NADPH levels were decreased, and the yields of 2.3-BDO were increased but the acetoin titer was decreased when compared with GDH solely overexpressed strains BM2, BM3, BM4 and BM5 (Tale 3).The strain BM12 (GDH under promoter P pac ) showed the highest titer of 2,3-BDO which was increased by 39.64% while the acetoin titer was decreased by 36.82 %, which suggested that excessive NADPH was used for the synthesis of 2,3-bdo, and more acetoin was catalyzed to 2,3-bdo.
When compared with the parental strain B.subtilis 168, it could be found that introduction of an e cient exogenous NADPH/NADP + regeneration system (co-overexpressing GDH and TDH) in strain BM12 increased 2,3-BDO titer by 14.01% and total amount of acetoin and 2,3-BDO by 3.05% while decreased the titers of NADH-dependent by-products lactate and ethanol by 12.82% and 23.65% , respectively.It indicated that we successfully redistributed the carbon ux to 2,3-BDO pathway by regulating cofactors regeneration systems in B. subtilis.
Redistributing the carbon ux to 2,3-BDO by overexpressing the transcriptional regulator ALsR and blocking the pathway to lactate ALsR is a LysR-type transcriptional regulator , which could regulate the alsSD operon, encoding the key enzyme (ALS and ALDC), catalyzing pyruvate to acetoin [28,29].To redistribute more carbon ux to 2,3-BDO pathway, ALsR under promoter P srfA [30] was overexpressed by integrated into the chromosome of BM12 at ldh(encoding lactate dehydrogenase , LdhA) locus, resulting in strain BM15.The multiple copies of alsr improved the activities of als (from 1.0 U/mg to 1.75U/mg) and aldc (from 0.51 U/mg to 0.75U/mg) by 75% and 47.05% and decreased the activity of LdhA by 89 % when compared with strain BM12.Shown in Table 3.
As shown in Figure 3A, the cell density has no obvious change among BM0, BM12 and BM15.However, the introduction of extra copies of alsR and disrupt of ldh in strain BM15 resulted in faster glucose consume rate, increased 2,3-BDO by 2.5% and decreased acetoin and lactate titer by 10.34% and 59.03% compared with strain BM12.Furthermore, when compared with parental strain B.subtilis 168, through all above metabolic engineering strategies in strain BM15, we shortened the fermentation time from 72 hours to 70 hours, increased 2, 3-BDO titer and total titer of acetoin and 2,3-BDO by 18.35% and , while decreased the titers of acetoin and NADH-dependent by-products lactate by 30.64% and 64.28% respectively(Figure3).

Discussion
As fossil fuels become increasingly scarce, bio-production of 2,3-BDO is attracting increasing attention [1].Generally, sugars are converted into pyruvate mainly from glycolysis before the target product 2,3-BD is formed [11].As known that pyruvates could be also produced via PPP.However, it was found that PPP is an ine cient synthetic pathway for pyruvate production from glucose in B. subtilis [19].GDH (encoding by gdh) is one of the key enzymes catalyzes the conversion of glucose to gluconate which enter PPP [19] .Thus, we tried to overexpress GDH in B. subtilis to enhance the metabolic e ciency of PPP and broaden the pathways from glucose to pyruvate.It was found that overproduction of GDH in B. subtilis increased NADPH levels and acetoin accumulation but decreased NADH levels and 2,3 -BDO production, and extended the duration.The disturbed levels of cofactors should be responsible for inhibited carbon utilization rate.Lower levels of NADH decreased NADH-dependent products 2,3-BDO, lactate and ethanol production.Insu ciency NADH availability inhibited transformation of acetoin to 2, 3-BDO, so acetoin accumulated more.
In 2,3-BDO biosynthesis pathway, it contains a series of redox reactions which participate in regulating the NADH/NAD + ratio in bacteria [14].Overproduction of GDH in B. subtilis disturbed NADH and NADPH levels and repressed 2,3-BDO fermentation.Thus, we need introduce an exogenous NADPH/NADP + regeneration system to rebalance cofactors regeneration system in B. subtilis and improve 2,3-BDO fermentation.NADPH-dependent 2.3-BDO dehydrogenase could be found in anaerobic microorganisms such as Clostridium beijerinckii [31] and Clostridium.autoethanogenum [21].Some cofactors metabolic engineering strategies have been employed to improve 2, 3-BDO production and repress byproducts production by manipulating NADH levels.To improve the NADH yield from NADPH in B. subtilis, Fu et al. introduced a transhydrogenase and successfully enhanced the 2,3-BDO titer by 13.6% [16].In previous study, we manipulated NADH levels in B. subtilis by introducing an extra NADH regeneration system (Formate dehydrogenase and formate) while simultaneously disrupting the NADH oxidase and ldh genes, which increased the 2,3-BDO titer by 25.5%, with a concomitant decrease acetoin (by 76.4%) and lactate (by 80.5%) accumulation [15].Inspired by these results, we tried to set up a NADPH/NADP + regeneration cycle to redistribute the carbon ux to 2,3-BDO with NADPH as cofactor.As it was expected, when a NADPH-dependent 2.3-BDO dehydrogenase was successfully co-overexpressed with GDH in B. subtilis, it signi cantly increased the 2,3-BDO production and sharply decreased acetoin and NADH-dependent by-products lactate and ethanol formation, and shortened the fermentation duration.It indicated that we successfully redistributed the carbon ux to 2,3-BDO pathway and improve the consumption rate of glucose by reconstructing the NADPH/NADP + regeneration systems in B. subtilis.
In bacterial metabolism, ethanol, acetoin, lactate, and other end-products are also produced during 2,3-BD fermentation [32].Therefore, 2,3-BD biosynthesis must compete with multiple pathways for the pyruvate and NADH resources, which remains prohibitively low for commercial production of 2,3-BDO.To improve the yield of 2,3-BDO, overproduction of key enzymes involved in the 2,3-BDO pathway and blocking the unwanted by-products pathways are all alternative strategies.ALsR acts as the regulatory gene of alsSD operon encoding ALS and ALDC, which is responsible for acetoin biosynthesis [28,29].Some researchers had tried to overexpressing ALS and ALDC to enhance the acetoin production.However, the higher activities of ALS and ALDC suppressed the cell growth, the acetoin yield was not signi cantly increased [23,33].Zhang et al. attempted to control ALS and ALDC by moderately enhancing the expression of AlsR, and successfully improved the yield of acetoin by 62.9% in B. subtilis [23].The deletion of adhE (encoding alcohol dehydrogenase) and ldh gene (encoding lactate dehydrogenase) could block the carbon ux toward ethanol and lactate biosynthesis increase the available NADH for 2,3-BD formation [34][35][36].In this study, to redistribute the carbon ux to 2,3-BDO ,we tried to regulate the expression level of ALsR using the promoter P srfA , which is self-induced and transcribes at the middle and late stage of fermentation [30].The fused gene fragment P srfA -ALsR was inserted into the location of ldh in the B. subtilis genome, which resulted in the recombinant strain in which ALsR was overproduced and ldh was disrupted simultaneously.It was found that the lacking of ldh and extra copies of ALsR in B. subtilis resulted in more carbon to ow into the 2, 3-BDO synthesis pathway, and increased the 2, 3-BDO titer by 18.35% and decreased the yields of by-products AC, lactate and ethanol by 22.03 %, 64.28 % and 25.80% respectively.The fermentation duration was shortened to 70 h.

Conclusion
In this study, we proposed an alternative strategy to regulate the carbon ux toward 2,3-BDO by introducing an exogenous NADPH/NADP + regeneration system, overexpressing ALsR under the promoter P srfA and disrupting ldh gene simultaneously.
The production of 2,3-BDO was increased, and the by-products production of acetoin, and lactate and ethanol were decreased signi cantly, which indicated that engineering of NADPH coenzyme cycles could improve 2,3-BDO production and suppress the by-products accumulation.

Bacterial strains, primers, and plasmids
All bacterial strains used are described in Table 1.All primers and plasmids used in this study are described in Table 2 Culture conditions Strains were cultured in Luria-Bertani medium at 37 °C on a rotary shaker at 180 rpm, if necessary, add 50 mg / L of Kana Magnesium, 100 mg/L ampicillin or 3% bleomycin into the medium.
For 2,3-BDO fermentation, the fermentation medium included 100 g/L glucose ,5 g/L yeast extract 20 g/L corn syru and 2 g/L urea, pH 6.8.The fermentation medium was sterilized at 121 °C for 20 min.The seed was cultured in the LB at 37 °C on a rotary shaker at 180 rpm for 12 h, 3 mL seed was inoculated into 50 mL fermentation medium at 180 rpm and 37 °C.

The construction plasmids and Strains
The promoter P bdhA and gene gdh was ampli ed from the B. subtilis 168 genome by the primers P1 and P2, P9 and P10 respectively, the promoters Ppac and Pspac were synthesized in Genewize company.It was obtained by PCR by primers P3 and P4, P5 and P6.Promoters P bdhA , P pac , P Spac , P HpaII and gdh genes connected together by overlap extension PCR(SOE PCR) [37], then insert to the site of EcoRI in pMA5.For the construction of pMA5-P HpaII -TDHRX (X was from 1to 5), the gene tbdh was synthesized in Genewize company, TDH, TDHR1, TDHR2, TDHR3, TDHR4, TDHR5, design with were obtained by PCR with primers p11 and p12, p13, p14, p15, p16, p17 respectively, and then ligated into the vector pMA5 at NdeI and BamHI restriction sites.For the pMA5-P HpaII -TDHR5-Ppac-gdh construction, rstly construct pMA5-P HpaII -TDHR5, then inserted gdh under different promoters insert to the site of EcoRI of pMA5.All ligation between genes and vector were used Clon Express II One Step Cloning Kit (Vazyme Biotech in NanJing, China).
Gene alsR and the promoter P srfA were ampli ed by PCR by primers P26 and P27, P24 and P25, respectively.The gene zeo was ampli ed from p7z6 by primers P18 and P19.ldhA-F (1000 bp upstream ldhA gene in genome) and ldhA-R (1000 bp downstream ldhA gene in genome) were obtained by primers P20 and P21, P22 and P23, respectively.Then, ldhA-R, ldhA-F,zeo, P srfA and alsR were connected together through SOE-PCR.
The method of transform plasmid or DNA fragments into Bacillus subtilis 168 reference the method described by Vojcic et al. [38] Enzyme assays To determine acetoin reductase (ACR) activity, bacteria was cultured overnight, the cells were collected by centrifugation for 5 min at 8000 rpm, and washed with 0.1 M pH 7.0 phosphate buffer for three times.ACR activity was assayed spectrophotometrically by monitoring the change in absorbance at 340 nm corresponding to the reduction of NADPH at 37 °C in a total volume of 1 mL 0.1 M phosphate buffer containing 0.2 mM NADPH and 50 mM acetoin.The enzyme activity unit was de ned that the amount of enzyme required to consume 1 μmol/L NADPH per minute.Total protein concentrations were determined according to the Bradford method.
To determine the activity of LdhA, the total volume of 1mL 0.1M phosphate buffer pH 7.2 containing 0.2 mM NADH and 10 mM sodium pyruvate was assayed spectrophotometrically by monitoring the change in absorbance at 340 nm corresponding to the reduction of NADH at 37 °C, the enzyme activity unit was de ned that the amount of enzyme required to consume 1 μmol/L NADH per minute.
The enzyme activities of ALS [39]and ALDC [40] asssys were performed according to published procedures.

Quantitative real-time PCR (qRT-PCR)
To determine the transcription abundance of tdh, cells cultured 8-10 h were harvested at room temperature and immediately frozen in liquid nitrogen.the total RNA of bacteria was extracted using Bacteria RNA Extraction Kit (Vazyme Biotech in NanJing, China), and then reverse-transcribed into cdna, which was used as the template for rt-pcr.Expression levels of different tbdh genes were measured with primers p28 and P29.The 16S rRNA gene was used as the internal standard with primers p30 and P31.
Transcriptional levels and activity of TDH in the recombinant strains.BM5, the control strain; BM6, strain with TDHR1; BM7, strain with TDHR2; BM8, strain with TDHR3; BM9, strain with TDHR4; BM10, strain with TDHR5; the 16S rRNA gene was used as the internal control gene to normalize the results.Error bars: Standard deviation (SD) (n =3).

Figures
Figures

Table 1
Strains used in this studyThe intracellular NADH and NADPH of BM1 BM2 BM3 BM4 were extracted at 72 h The intracellular NADH and NADPH of other strains were extracted at 60 h.