Article Application of a Pyruvate-Producing Strain LAFCPCPt-accBC-aceE: A Case Study for d-Lactate Production

Keisuke Wada 1,2 , Tatsuya Fujii 1,*, Hiroyuki Inoue 1, Hironaga Akita 1, Tomotake Morita 2 and Akinori Matsushika 1,3

1 Research Institute for Sustainable Chemistry (RISC), National Institute of Advanced Industrial Science and Technology (AIST), 3-11-32 Kagamiyama, Higashi-hiroshima, Hiroshima 739-0046, Japan; [email protected] (K.W.); [email protected] (H.I.); [email protected] (H.A.); [email protected] (A.M.) 2 Research Institute for Sustainable Chemistry (RISC), National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba Central 5-2, 1-1-1 Higashi, Tsukuba, Ibaraki 305-8565, Japan; [email protected] 3 Graduate School of Integrated Science for Life, Hiroshima University, 1-3-1 Kagamiyama, Higashi-hiroshima, Hiroshima 739-8530, Japan * Correspondence: [email protected]; Tel.: +81-82-493-6843

 Received: 11 June 2020; Accepted: 16 July 2020; Published: 17 July 2020 

Abstract: Pyruvate, a potential precursor of various chemicals, is one of the fundamental chemicals produced by the fermentation process. We previously reported a pyruvate-producing Escherichia coli strain LAFCPCPt-accBC-aceE (PYR) that has the potential to be applied to the industrial production of pyruvate. In this study, the availability of the PYR strain for the production of pyruvate-derivative chemicals was evaluated using a d-lactate-producing strain (LAC) based on the PYR strain. The LAC strain expresses a d--encoding gene from Lactobacillus bulgaricus under the control of a T7 expression system. The d-lactate productivity of the LAC strain was further improved by limiting aeration and changing the induction period for the expression of d-lactate dehydrogenase-encoding gene expression. Under combined conditions, the LAC strain produced d-lactate at 21.7 1.4 g L 1, which was compatible with the pyruvate production by the PYR strain ± · − (26.1 0.9 g L 1). These results suggest that we have succeeded in the effective conversion of pyruvate ± · − to d-lactate in the LAC strain, demonstrating the wide versatility of the parental PYR strain as basal strain for various chemicals production.

Keywords: Escherichia coli; pyruvate; fermentation; d-Lactate; NADH/NAD+ ratio

1. Introduction Pyruvate is one of the key metabolites in the central of all known organisms, and has been studied extensively for industrial use. The typical example of the use of pyruvate itself is the compound’s use in human health, for instance, in protecting cells against oxidative stress [1]. In addition, pyruvate is used as a precursor to various other chemicals, such as amino acids [2], alcohols [3], plastic monomers [4], and pharmaceuticals [5–7] (Figure1). Three methods of pyruvate production are known: chemical synthesis, fermentation, and enzymatic production [2]. Among the three, the fermentation method is known to have the lowest costs of raw materials, and commercial pyruvate production by fermentation has been developed in various organisms [2,8–10]. These facts indicate that it is possible to construct various chemical-producing strains based on a pyruvate-producing strains.

Fermentation 2020, 6, 70; doi:10.3390/fermentation6030070 www.mdpi.com/journal/fermentation Fermentation 2020, 6, x FOR PEER REVIEW 2 of 10 indicate that it is possible to construct various chemical-producing strains based on a pyruvate- Fermentation 2020, 6, 70 2 of 10 producing strains.

Figure 1. Derivatives of pyruvate. IPP and DMAPP indicate isopentenyl diphosphate and dimethylallyl Figure 1. Derivatives of pyruvate. IPP and DMAPP indicate isopentenyl diphosphate and pyrophosphate, respectively. dimethylallyl pyrophosphate, respectively. To enhance the productivity by strains of various chemicals, including derivatives of pyruvate, a rangeTo ofenhance metabolic the productivity modifications by of strains the producer of variou microbess chemicals, have beenincluding attempted. derivatives The regulationof pyruvate, of intracellulara range of metabolic balance modifications is one of theof the key producer strategies, microbes given that have cofactors been NAD(P)attempted.+ and The NAD(P)H regulation play of + importantintracellular roles redox in microbial balance metabolismis one of the [ 11key]. Forstrategies, example, given the inactivationthat cofactors of aldehydeNAD(P) dehydrogenaseand NAD(P)H toplay prevent important competition roles forin NADHmicrobial with metabolism -forming [11]. reactionsFor example, improved the 1,3-propanediolinactivation of production aldehyde withdehydrogenaseKlebsiella pneumoniae to preventby 2-foldcompetition [12]. In for another NADH example, with ethanol-forming the aeration control reactions to prevent improved competition 1,3- forpropanediol NADH with production the respiratory with Klebsiella chain improved pneumoniaeL-valine by 2-fold production [12]. In with anotherCorynebacterium example, the glutamicum aeration bycontrol 20-fold to prevent [13]. Therefore, competition the regulation for NADH of with intracellular the respiratory redox balance chain improved is important L-valine for the production microbial productionwith Corynebacterium of chemicals, glutamicum although by the 20-fold specific [13]. modifications Therefore, the needed regulation depend of onintracellular the production redox conditions,balance is important including for the the nature microbial of the hostproduction strains andof chemicals, the targeted although products. the specific modifications neededIn adepend previous on the report, production we described conditions, the including construction the nature of a pyruvate-producing of the host strains andE. the coli targetedstrain, LAFCPCPt-accBC-aceEproducts. (designated here as PYR) [14]. The genes accBC and aceE, encoding acetyl-CoA carboxylaseIn a previous and pyruvate report, dehydrogenase, we described respectively,the construction are known of a topyruvate-producing be essential for E. growth coli strain, when utilizingLAFCPCPt-accBC-aceE as the sole (designated carbon source here [15 as]. PYR) Hence, [14]. in the The PYR genes strain, accBC the expressionand aceE, encoding of accBC and acetyl-aceE wasCoA controlled carboxylase by and a doxycycline-inducible , system respecti [14,16vely,]. The are silencing known ofto beaccBC essentialand aceE for cellexpression growth usingwhen thisutilizing regulatory glucose system as the was sole an carbon effective source mechanism [15]. Hence, for inducing in the PYR pyruvate strain, production the expression [14,15 of]. Furthermore,accBC and aceE in was the controlled PYR strain, by severala doxycycline-induci genes were disruptedble system to [14,16]. enhance The pyruvate silencing productivity,of accBC and withaceE targetsexpression including: using this (i) biosynthetic regulatory system genes involved was an ineffective the production mechanism of ethanol, for inducing lactate, pyruvate , andproduction formate; [14,15]. and (ii) Furthermore,cra, a gene thatin the encodes PYR strain a global, several transcription genes were regulator disrupted (Figure to enhance2). In pyruvate simple batchproductivity, culture, thewith PYR targets strain including: showed high (i) biosynthet pyruvate productivityic genes involved (26.1 in0.9 the g L production1), demonstrating of ethanol, 56% ± · − elactate,fficiency acetate, per consumed and formate; glucose and when (ii) cra glucose, a gene was that used encodes as the a global sole carbon transcription source [ 14regulator]. These (Figure results −1 indicated2). In simple that PYRbatch is aculture, promising the foundationPYR strain strain showed for thehigh production pyruvate of productivity pyruvate derivatives. (26.1 ± 0.9 As g·L a first), exampledemonstrating of the application56% efficiency of the per PYR consumed strain, a d -lactate-producingglucose when glucose strain was (designated used as herethe assole old-LAC) carbon wassource constructed [14]. These by results introducing indicated a Lactobacillus that PYR is bulgaricum a promisinggene foundation encoding strain NAD+ for-dependent the productiond-lactate of dehydrogenasepyruvate derivatives. (DLDH) As into a first the example PYR background of the application [17]. The d of-lactate the PYR production strain, a pathwayD-lactate-producing was shown strain (designated here as old-LAC) was constructed by introducing a Lactobacillus bulgaricum gene1 in Figure2. However, the d-lactate production of the old-LAC strain was low level (1.1 0.0 g L− ) encoding NAD+-dependent D-lactate dehydrogenase (DLDH) into the1 PYR background [17].± The· D- compared with pyruvate production by the PYR strain (26.1 0.9 g L− ), implying that the expression lactate production pathway was shown in Figure 2. However,± the· D-lactate production of the old- of DLHD in the old-LAC strain was insufficient. These results indicate that there is room for further LAC strain was low level (1.1 ± 0.0 g·L−1) compared with pyruvate production by the PYR strain (26.1 improvement of the d-lactate production by the PYR-derived strain. ± 0.9 g·L−1), implying that the expression of DLHD in the old-LAC strain was insufficient. These results indicate that there is room for further improvement of the D-lactate production by the PYR-derived strain.

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Figure 2. Schematic of the d-lactate-producing (LAC) strain. The cross-marks denote Figure 2. Schematic metabolic pathway of the D-lactate-producing (LAC) strain. The cross-marks gene inactivation. d-lactate production (blue) competes with respiration (green) for NADH utilization. denote gene inactivation. D-lactate production (blue) competes with respiration (green) for NADH Theutilization. purpose of this study was to evaluate the availability of the PYR strain for the production of pyruvate-derivative chemicals by using a new d-lactate-producing strain (LAC) based on the PYR The purpose of this study was to evaluate the availability of the PYR strain for the production strain. The results of our study demonstrate the utility of the PYR strain for the development of of pyruvate-derivative chemicals by using a new D-lactate-producing strain (LAC) based on the PYR fermentation processes of pyruvate-based chemicals production. strain. The results of our study demonstrate the utility of the PYR strain for the development of 2.fermentation Materials and processes Methods of pyruvate-based chemicals production.

2.1.2. Materials Construction and ofMethods Bacterial Strains and Plasmids

2.1.1.2.1. Construction Plasmids of Bacterial Strains and Plasmids E. coli HIT-DH5α (SciTrove, Tokyo, Japan) was used for DNA manipulation. Ampicillin 2.1.1. Plasmids (100 mg L 1) and kanamycin (20 mg L 1) were added as necessary. In order to construct the expression · − · − vectorE. containingcoli HIT-DH5 theαL. (SciTrove, bulgaricus Tokyo,gene encoding Japan) was NAD used+-dependent for DNA DLDHmanipulation. under control Ampicillin of the (100 T7 promoter,mg·L−1) and the kanamycinDLDH in pMALc5x(20 mg·L−1/)D- wereLDH added [17] was as necessary. excised with In order NdeI andto construct BamHI andthe expression subcloned betweenvector containing the NdeI andthe L. BamHI bulgaricus sites ofgene pETIK encoding (BioDynamics NAD+-dependent Laboratory DLDH Inc., Tokyo, under Japan) control to of generate the T7 pETIK-promoter,DLDH the. DLDH in pMALc5x/D-LDH [17] was excised with NdeI and BamHI and subcloned between the NdeI and BamHI sites of pETIK (BioDynamics Laboratory Inc., Tokyo, Japan) to generate 2.1.2.pETIK- BacterialDLDH. Strains The LAC strain was constructed based on the PYR strain [14]. The λDE3 lysogenization kit (Novagen,2.1.2. Bacterial Madison, Strains WI, USA) was employed to introduce a T7-driven expression system into the chromosomeThe LAC strain of the was PYR constructed strain according based on to the kitPYR manufacture’s strain [14]. The protocol. λDE3 Thelysogenization insertion into kit the(Novagen, PYR strain Madison, chromosome WI, USA) of was the employed gene encoding to introduce T7 RNA a T7-driven polymerase expression was confirmed system into by the PCR-basedchromosome screening of the PYR of strain candidate according strains to usingthe kit primersmanufacture’s 50-ATGAACACGATTAACATCGC-3 protocol. The insertion into the 0PYRand 5strain0-TTACGCGAACGCGAAGTC-3 chromosome of the gene encoding0. The selected T7 RNA strain polymerase (designated was PYR(DE3)) confirmed then by was the transformed PCR-based withscreening pETIK of (the candidate empty vector) strains or pETIK-using primersDLDH, yielding 5′ -ATGAACACGATTAACATCGC-3 strains designated NULL or LAC,′ respectivelyand 5′ - (TableTTACGCGAACGCGAAGTC-31). The NULL strain was used′. The as aselected control. strain (designated PYR(DE3)) then was transformed with pETIK (the empty vector) or pETIK-DLDH, yielding strains designated NULL or LAC, respectively (Table 1). The NULL strain was used as a control.

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Table 1. E. coli strains used in this study.

Strains Relevant Descriptions 1 References Derived of the MG1655; ∆ldhA ∆adhE ∆pflB ∆(ackA-pta) ∆poxB ∆cra PYR [14] PaccBC::Ptet-accBC PaceEF::Ptet-aceE old-LAC Derived of the PYR strain; [pMAL-c5X-DLDH][17] PYR(DE3) Derived of the PYR strain; λ(DE3) This study NULL Derived of the PYR(DE3) strain; [pETIK] 1 This study LAC Derived of the PYR(DE3) strain; [pETIK-DLDH] This study 1 [] indicates plasmid-carrier state.

2.2. Culture Conditions for d-Lactate Production All E. coli strains were pre-cultured for 12 h at 37 C with 250 rpm in LB medium (5 g L 1 ◦ · − extract, 10 g L 1 tryptone, and 10 g L 1 NaCl). Main cultures were generated by an inoculation · − · − of pre-cultures into N5G medium (1 g L 1 K HPO , 10 g L 1 (NH ) SO , 2 g L 1 NaCl, 0.25 g L 1 · − 2 4 · − 4 2 4 · − · − MgSO 7H O, 15 mg L 1 CaCl 2H O, 60 g L 1 glucose, and 0.2 g L 1 Adekanol LG-294 as an antifoam 4· 2 · − 2· 2 · − · − agent) at 600 nm (OD600) of 0.05. Culturing for d-lactate production was performed by growth for 72 h in a Bio Jr. 8 small-scale bioreactor (ABLE & Biott, Tokyo, Japan). The basal conditions were as follows: volume, 80 mL; temperature, 35 C; aeration, agitation at 1800 rpm and airflow at 150 mL min 1; pH 5.7 ◦ · − (as adjusted using 4 M NaOH); induction point, 12 h by 0.1 mM isopropyl β-D-thiogalactopyranoside (IPTG). Dry cell weight was calculated using a conversion coefficient of 0.3 gDCW L 1 OD 1 [18]. · − 600− 2.3. Quantification of Metabolites

2.3.1. Extracellular Metabolites The concentrations of extracellular metabolites (glucose, pyruvate, DL-lactate, formate, acetate, and ethanol) in the filtered culture supernatants were determined using an high-performance liquid chromatography (HPLC) system (JASCO, Tokyo, Japan) equipped with an Aminex HPX-87H column (Bio-Rad, Hercules, CA, USA), a UV-2070 Plus UV/vis detector, and a RI-2031 Plus refractive index detector. The chromatographic conditions were as follows: mobile phase, 1.5 mM H2SO4; flow rate, 0.6 mL min 1; column oven temperature, 65 C. The purity check of the produced d-lactate was · − ◦ performed using an HPLC system equipped with a CHIRALPAK MA(+) column (Daicel, Osaka, Japan). The chromatographic conditions for purity check were as follows: mobile phase, 0.5 mM CuSO4; flow rate, 0.4 mL min 1; column oven temperature, 30 C. · − ◦ 2.3.2. Intracellular NAD+ and NADH The intracellular NAD+ and NADH were extracted and quantified using an EnzyChrom™ NAD+/NADH Assay Kit (BioAssay Systems, Hayward, CA, USA) and a Synergy HTX fluorescence plate reader (BioTek, Winooski, VT, USA) according to the kit manufacture’s protocol.

2.4. Assays of DLDH The old-LAC and the LAC strain cells under the basal conditions were collected at 36 h. All procedures after cell collection were performed on ice. The collected cells were washed twice with 9 g L 1 NaCl, resuspended with 150 mM potassium phosphate (pH 6.5), homogenized by · − ultrasonication, and centrifuged. The resultant supernatants were used as crude extract. The protein concentration in the crude extract was determined by using the Bradford protein assay kit (Bio-Rad, CA, USA). The DLDH activity was measured spectrophotometrically at 25 ◦C by monitoring NADH consumption at 340 nm with GeneQuant 1300 (GE Healthcare, Chicago, IL, USA). The reaction mixture (1 mL) contained 150 mM potassium phosphate (pH 6.5), 30 mM pyruvate, 2.5 mM MgCl2, 0.3 mM NADH, and the crude extract. The reaction was initiated by the addition of the crude extract. The extinction coefficient of NADH was 6.22 mM 1 cm 1. − · − FermentationFermentation 20202020,, 66,, x 70 FOR PEER REVIEW 55 of of 10 10

3. Results 3. Results 3.1. Culture Profiles of the LAC Strain under Basal Conditions 3.1. Culture Profiles of the LAC Strain under Basal Conditions In our previous study, the relatively low production of D-lactate by the old-LAC strain suggested In our previous study, the relatively low production of d-lactate by the old-LAC strain suggested the possibility that the expression of DLDH in the old-LAC strain was limiting. The previous report the possibility that the expression of DLDH in the old-LAC strain was limiting. The previous report showed that the T7 promoter was more effective for the targeted protein production in E. coli [19]. showed that the T7 promoter was more effective for the targeted protein production in E. coli [19]. Hence, we replaced the tac promoter originally used to drive DLDH expression on pMAL-c5X with Hence, we replaced the tac promoter originally used to drive DLDH expression on pMAL-c5X with the the T7 promoter on pETIK, resulting in the LAC strain (Table 1). T7 promoter on pETIK, resulting in the LAC strain (Table1). The culture profiles of the NULL (used as control because of same genetic background with the The culture profiles of the NULL (used as control because of same genetic background with LAC strain except for DLDH) and LAC strains under the basal conditions used in the previous study the LAC strain except for DLDH) and LAC strains under the basal conditions used in the previous [14] are shown in Figure 3. The final cell density and glucose consumption of the LAC strain were study [14] are shown in Figure3. The final cell density and glucose consumption of the LAC strain −1 −1 −1 −1 1.2-fold (=4.23 gDCW·L /3.64 gDCW·L1 ) and 1.8-fold1 (=43.83 g·L /24.32 g·L1 ) those 1of the NULL were 1.2-fold (=4.23 gDCW L− /3.64 gDCW L− ) and 1.8-fold (=43.83 g L− /24.32 g L− ) those of the strain, respectively. The pyruvate· produced ·by the NULL strain is lower· than that by· the PYR strain NULL strain, respectively. The pyruvate produced by the NULL strain is lower than that by the PYR [14]. The unexpected product from multi-cloning site of pETIK might be affected to pyruvate strain [14]. The unexpected product from multi-cloning site of pETIK might be affected to pyruvate production by the NULL strain. As expected, D-lactate production was observed only in the LAC production by the NULL strain. As expected, d-lactate production was observed only in the LAC strain. strain. The D-lactate produced by the LAC strain demonstrated over 99% optical purity (data not The d-lactate produced by the LAC strain demonstrated over 99% optical purity (data not shown). −1 shown). The highest concentration of D-lactate produced by the LAC strain was 4.11 ± 0.4 g·L at 36 h, The highest concentration of d-lactate produced by the LAC strain was 4.1 0.4 g L− at 36 h, which was which was approximately 9.0-fold (=4.07 g·L−1/0.45 g·L−1) that of the old-LAC,± at· 36 h [17]. The DLDH approximately 9.0-fold (=4.07 g L 1/0.45 g L 1) that of the old-LAC, at 36 h [17]. The DLDH activity − − −1 −1 −1 −1 activity at 36 h of the LAC strain· was 33-fold· (=292.40 µmol·mg1 1·min /9.01 µmol·mg1 ·min1 ) that of at 36 h of the LAC strain was 33-fold (=292.40 µmol mg− min− /9.01 µmol mg− min− ) that of the the old-LAC strain. From these results, it seemed that· the DLDH· activity was· correlated· with the D- old-LAC strain. From these results, it seemed that the DLDH activity was correlated with the d-lactate lactate productivity. In addition, the amount of glucose consumed through until 72 h by the LAC productivity. In addition, the amount of glucose consumed through until 72 h by the LAC strain was −1 −1 strain was 11-fold (= 143.83 g·L /3.991 g·L ) that of the old-LAC strain [17]. Most notably, however, the 11-fold (= 43.83 g L− /3.99 g L− ) that of the old-LAC strain [17]. Most notably, however, the d-lactate D-lactate concentration· in the· culture medium decreased gradually between 36 h and 72 h (from 4.1 concentration in the culture medium decreased gradually between 36 h and 72 h (from 4.1 0.4 g L 1 −1 −1 − ± 0.4 g·L to 0.2 1± 0.1 g·L , respectively), accompanied by an increase in the pyruvate concentration± · 1 to 0.2 0.1 g L− , respectively), accompanied by an increase in the pyruvate concentration (0 0 g L− (0 ± 0 g·L± −1 to· 16.1 ± 1.4 g·L−1, respectively). These data suggest that the D-lactate that was produced± · to 16.1 1.4 g L 1, respectively). These data suggest that the d-lactate that was produced after 36 h after 36 ±h was ·subsequently− reconverted to pyruvate. was subsequently reconverted to pyruvate.

(a) (b) Figure 3. Growth characteristics of the NULL and LAC strains grown under the basal conditions. Figure 3. Growth characteristics of the NULL and LAC strains grown under the basal conditions. The The (a) NULL and (b) LAC strains were cultured in N5G medium under the basal conditions [14]. (a) NULL and (b) LAC strains were cultured in N5G medium under the 1basal conditions [14]. Parameters: temperature, 35 ◦C; aeration, 1800 rpm (agitation) and 150 mL min− (airflow rate); pH, 5.7; Parameters: temperature, 35 °C; aeration, 1800 rpm (agitation) and 150 mL·min· −1 (airflow rate); pH, induction point, 12 h (vertical dashed line). Symbols: filled circles, OD600; squares, glucose; triangles, 5.7; induction point, 12 h (vertical dashed line). Symbols: filled circles, OD600; squares, glucose; acetate; red diamonds, pyruvate; blue diamonds, d-lactate. Data shown are the mean SD (n = 3). triangles, acetate; red diamonds, pyruvate; blue diamonds, D-lactate. Data shown are ±the mean ± SD 3.2. Regulation(n = 3). of Aeration Improves d-Lactate Production The production of pyruvate from d-lactate was expected to be accompanied by NADH generation 3.2. Regulation of Aeration Improves D-Lactate Production (d-lactate + NAD+ => pyruvate + NADH). The generated NADH presumably then would be used for OThe2-respiration, production given of pyruvate that the basalfrom conditionsD-lactate was were expected aerobic (Figureto be 2accompanied). Hence, to preventby NADH the + generationO2-respiration, (D-lactate we limited + NAD the => aeration pyruvate after + NADH). 36 h by The decreasing generated the NADH agitation presumably and air-flow then rate would from be used for O2-respiration, given that the basal conditions were aerobic (Figure 2). Hence, to prevent

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the O2-respiration, we limited the aeration after 36 h by decreasing the agitation and air-flow rate 1800from rpm 1800 and rpm 150 mL andmin 1501 mL·min(respectively)−1 (respectively) to 300 rpm to and 300 0 mL rpmmin and1 (termed0 mL·min air-regulated−1 (termed conditions)air-regulated · − · − (Figureconditions)4). (Figure 4).

(b) (a)

Figure 4. d-Lactate production under air-regulated conditions. The values of aeration parameters Figure 4. D-Lactate production under air-regulated conditions. The values of aeration parameters were changed at 36 h (central vertical dashed line) from 1800 rpm and 150 mL min 1 to 300 rpm and were changed at 36 h (central vertical dashed line) from 1800 rpm and 150 ·mL·min− −1 to 300 rpm and 0 mL min 1, respectively. (a) The culture profile of the LAC strain and (b) NADH/NAD+ ratio for 0 mL·min· − −1, respectively. (a) The culture profile of the LAC strain and (b) NADH/NAD+ ratio for cultures grown under the basal (white) and the air-regulated (gray) conditions. Symbols: filled circles, cultures grown under the basal (white) and the air-regulated (gray) conditions. Symbols: filled circles, OD600; squares, glucose; triangles, acetate; red diamonds, pyruvate; blue diamonds, d-lactate. The data OD600; squares, glucose; triangles, acetate; red diamonds, pyruvate; blue diamonds, D-lactate. The shown are the mean SD at 72 h (n = 3); values in (b) are at 72 h. data shown are the± mean ± SD at 72 h (n = 3); values in (b) are at 72 h. The culture profiles of LAC strain until 36 h were similar to those for cells growing under the basalThe conditions culture profiles (Figures of 3LACb and strain4a). However,until 36 h thewere cell similar density to those reached for acells plateau growing under under the the basal conditions (Figures 3b and 4a). However, the cell1 density reached1 a plateau under the air- air-regulated conditions and was 3.2-fold (=4.23 gDCW L− /1.28 gDCW L− ) lower than that under the · −1 · −1 basalregulated conditions conditions at 72 h. and Onthe was other 3.2-fold hand, (=4.23 the concentration gDCW·L /1.28 of d gDCW·L-lactate increased) lower graduallythan that throughunder the basal conditions at 72 h. On1 the other hand, the concentration1 of D-lactate1 increased gradually through 72 h, reaching 12.1 1.6 g L− , which was 53-fold (=12.1 g L− /0.23 g L− ) that obtained under the basal 72 h, reaching 12.1± ± 1.6· g·L−1, which was 53-fold (=12.1· g·L−1/0.23· g·L−1) that obtained under the basal1 conditions. The pyruvate production was lower under the air-regulated conditions (0.7 0.6 g L− ) ± −1 comparedconditions. to that The obtained pyruvate under production the basal was conditions. lower under These the data air-regulated indicated that conditions changing (0.7 the ± aeration 0.6 g L ) parameterscompared at to 36 that h was obtained an effective under method the basal for cond enhancingitions. dThese-lactate data production. indicated Thethat intracellular changing the NADHaeration/NAD parameters+ ratio at 72 at h 36 under h was the air-regulatedan effective conditionsmethod for was enhancing 1.3-fold (=D1.69-lactate/1.29) production. that obtained The + underintracellular the basal NADH/NAD conditions (Figure ratio4b), at suggesting72 h under that the theair-regulated decreased aerationconditions provided was 1.3-fold an intracellular (=1.69/1.29) redoxthat stateobtained suitable under for the the basal conversion conditions from (Figure pyruvate 4b), to suggestingd-lactate [ 20that]. the decreased aeration provided an intracellular redox state suitable for the conversion from pyruvate to D-lactate [20]. 3.3. Improvement of d-Lactate Production and Cell Density by Pre-Induction of DLDH 3.3. Improvement of D-Lactate Production and Cell Density by Pre-Induction of DLDH To further increase the productivity, the influence of the induction period of DLDH was investigated. When 0.1To mMfurther IPTG increase was included the productivity, in the pre-culture the influence medium, of dthe-lactate induction production period at of 72 DLDH h under was D 1 1 theinvestigated. air-regulated When conditions 0.1 mM (termed IPTG was combined included conditions) in the pre-culture was 1.8-fold medium, (=21.72 -lactate g L− / 12.09production g L− ) at that72 obtainedh under the from air-regulated a main culture conditions initiated (term usinged combined IPTG-naïve conditions) pre-culture was (Figure1.8-fold5 ).(=21.72 In addition, g L−1/12.09 −1 1 1 theg glucoseL ) that consumptionobtained from under a main the culture combined initiated conditions using IPTG-naïve was 2.1-fold pre-culture (=35.0 g (Figure L− /16.4 5). g In L− addition,) that underthe glucose the air-regulated consumption conditions. under Thethe maximumcombined cellconditions density was under 2.1-fold the combined (=35.0 g conditions L−1/16.4 g atL− 361) that h 1 1 wasunder 2.4-fold the (air-regulated=2.78 gDCW conditions. L− /1.16 gDCW The maximum L− ) that under cell density the air-regulated under the combined conditions. conditions The specific at 36 d-lactateh was 2.4-fold production (=2.78 rates gDCW after L− 361/1.16 h by gDCW the LAC L−1) strainthat under under the the air-regulat air-regulateded conditions. and the combinedThe specific conditionsD-lactate wereproduction same level rates (Figure after 36 S1). h by These the dataLAC suggested strain under that the the air-regulated increase of the and cell the density combined by pre-inductionconditions were of DLDH samehad level a positive(Figure S1). effect These on d -lactatedata suggested production. that the increase of the cell density by pre-induction of DLDH had a positive effect on D-lactate production.

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Figure 5. 5. CultureCulture profiles profiles of of the the LAC LAC strain strain under under the the combined combined conditions. conditions IPTG. IPTG was was added added to the to pre-culturethe pre-culture at 0 h at and 0 h to and the tomain the culture main culture at 12 h. at The 12 values h. The of values the aeration of the aerationparameters parameters were changed were 1 1 atchanged 36 h from at 36 1800 h from rpm 1800 and rpm 150 andmL min 150 mL−1 tomin 300− rpmto 300and rpm 0 mL and min 0 mL−1, respectively. min− , respectively. Symbols: Symbols: circles, d ODcircles,600; squares, OD600; squares, glucose; glucose; triangles, triangles, acetate; acetate;red diamonds, red diamonds, pyruvate; pyruvate; blue diamonds, blue diamonds, D-lactate.-lactate. Data Data shown are the mean SD (n = 3). shown are the mean ± SD (±n = 3). 3.4. Summary of Culture Profiles 3.4. Summary of Culture Profiles The values of the culture profiles are summarized in Table2. The final concentration of d-lactate The values1 of the culture profiles are summarized in Table 2. The final concentration of D-lactate (21.7 1.4 g L− ) obtained with the LAC strain under the combined conditions approached that of −1 (21.7 ± 1.4 g L ) obtained1 with the LAC strain under the combined conditions approached that of pyruvate (26.1 0.9 g L− ) obtained with the PYR strain under basal conditions [14]. In addition, −1 pyruvate (26.1 ±± 0.9 g L ) obtained with the PYR strain under 1basal conditions1 [14]. In addition, the the d-lactate productivity was increased 20-fold (=21.72 g L− /1.07 g L− ) compared to that in our D-lactate productivity was increased 20-fold (=21.72 g L−1/1.07 g L−1) compared to that in our previous previous study using the old-LAC strain [17]. The yield of d-lactate production per consumed glucose study using the old-LAC strain [17]. The yield of D-lactate production1 1 per consumed1 glucose1 under under the combined conditions was 2.3-fold (=(21.72 g L− /35.0 g L− )/(1.07 g L− /3.99 g L− )) that the combined conditions was 2.3-fold (=(21.72 g L−1/35.0 g L−1)/(1.07 g L−1/3.99 g L−1)) that obtained in obtained in the previous study [17]. These results suggested that we succeeded in our goal of efficiently the previous study [17]. These results suggested that we succeeded in our goal of efficiently producing d-lactate as a pyruvate derivative by combining the construction of a new strain and changes producing D-lactate as a pyruvate derivative by combining the construction of a new strain and in culture conditions. changes in culture conditions. Table 2. Summary of culture profiles at 72 h. Table 2. Summary of culture profiles at 72 h. Consumed Glucose Produced d-Lactate Produced Pyruvate Yields 1 Strains Conditions 1 1 1 1 References (g L− ) (gProducedL− ) Produced(g L− ) (g g− ) Consumed· Glucose · · Yields· 1 StrainsPYR Conditions Basal 48.0 4.7 DN-Lactate/A 2 Pyruvate26.1 0.9 54 2References [14] (g·L± −1) ± (g·g−±1) old-LAC Basal 3 4.0 1.9 1.1 0.0−1 · N/−A1 33 12 [17] ± (g·L± ) (g L ) ± LAC Basal 43.8 4.7 0.2 0.1 16.1 1.4 1 0 This study PYR Basal 48.0± ± 4.7 N/A± 2 26.1 ±± 0.9 54 ±± 2 [14] LAC Air-regulated 16.4 2.6 12.1 1.6 0.7 0.6 74 8 This study 3 ± ± ± ± old-LACLAC CombinedBasal 35.0 4.00.8 ± 1.9 21.7 1.1 ±1.4 0.0 0.1 N/A0.0 33 62± 123 This [17] study LAC Basal 43.8± ± 4.7 0.2± ± 0.1 16.1 ±± 1.4 1 ± 0± This study 1 Produced pyruvate by PYR strain or d-lactate by LAC series strains per consumed glucose. 2 N/A indicates not 3 1 LACapplicable. Air-regulatedThe pH value and the 16.4 concentration ± 2.6 of NH 12.14Cl were ± 1.6 set to 6.0 and 0.7 20 ± g0.6 L− , respectively. 74 ± 8 This study LAC Combined 35.0 ± 0.8 21.7 ± 1.4 0.1 ± 0.0 62 ± 3 This study 4. Discussion1 Produced pyruvate by PYR strain or D-lactate by LAC series strains per consumed glucose. 2 N/A indicates not applicable. 3 The pH value and the concentration of NH4Cl were set to 6.0 and 20 g L−1, In this study, to evaluate the availability of the PYR strain for the production of pyruvate-derivative respectively. chemicals, we investigated the d-lactate production ability of the LAC strain, which is based on the 4.PYR Discussion strain. The maximum d-lactate productivity in this study was 20-fold greater than that obtained in our previous study [17]. Furthermore, the d-lactate productivity of the LAC strain (21.7 1.4 g L 1) ± · − was comparableIn this study, with to theevaluate pyruvate the productivity availability ofof the the PYR PYR strain strain (26.1 for the0.9 gproductionL 1)[14], indicating of pyruvate- that ± · − derivativemost of the chemicals, produced we pyruvate investigated was converted the D-lactate to d-lactate. production Since ability lactate of is the a monomer LAC strain, of poly-lactic which is basedacid, which on the is PYR one strain. of the biodegradableThe maximum andD-lactate biocompatible productivity plastic in this polyesters, study was it may 20-fold contribute greater to than the thatproduction obtained of ind-lactate our previous by the LACstudy strain [17]. becauseFurthermore, of low the by-product D-lactate production.productivity of the LAC strain −1 −1 (21.7 This± 1.4 work g·L ) represents was comparable the first with study the demonstrating pyruvate produc the versatilitytivity of the ofour PYR pyruvate-producing strain (26.1 ± 0.9 g·L PYR) [14],strain. indicating To enhance that the mostd-lactate of the productivity produced pyruvate of the LAC was strain, converted we modified to D-lactate. the culture Since conditions lactate is ina monomertwo steps. of First, poly-lactic aeration acid, was which limited is afterone of 36 the h of biodegradable cultivation (Figure and biocompatible4a). Our previous plastic study polyesters, showed itthat may pyruvate contribute production to the by production the PYR strain of D was-lactate favored by underthe LAC more strain aerobic because conditions of low [14 ],by-product suggesting production. that the NADH generated in glycolysis was consumed by O2-respiration to maintain intracellular redox This work represents the first study demonstrating the versatility of our pyruvate-producing PYR strain. To enhance the D-lactate productivity of the LAC strain, we modified the culture

Fermentation 2020, 6, 70 8 of 10 homeostasis. Other pyruvate production studies have also employed aerobic conditions, irrespective of the bacterial species [8–10]. On the other hand, a shift to d-lactate production was expected to create competition for NADH consumption between the pyruvate to d-lactate reaction and O2-respiration. Therefore, the limiting aeration after 36 h of cultivation was expected to favor d-lactate production over O2-respiration. Second, we changed the induction period of DLDH expression (Figure5). In the PYR strain, the NADH generated by glycolysis was not consumed by O2-respiration during the early phase of cultivation because the expression of the pyruvate dehydrogenase gene has been suppressed at early time points [14]. In fact, initial cell growth by the PYR strain was observed to be repressed until 12 h [14]. On the other hand, the initial cell growth of the LAC strain was enhanced by the expression of DLDH starting in the pre-culture (Figure5), suggesting that the NADH produced in the early-phase culturing was consumed by DLDH. However, glucose remained in the culture at 72 h of growth under the combined conditions (Figure5). In addition, specific d-lactate production rates by the LAC strain in the air-regulated and the combined conditions were same level (Figure S1). These results implied that d-lactate production by the LAC strain might be improved by further increases in DLDH activity. The LAC strain exhibited superior d-lactate productivity in the simple batch culture upon the modification of the culture conditions from those of our previous study [14]. Since the genes encoding related to the production of major organic acids (pta-ackA and poxB, acetate; pflB, formate; ldhA, d-lactate; adhE, ethanol) are deleted in the PYR strain, high-purity fermentation production is expected following the introduction of genes encoding enzymes for the synthesis of target products, as demonstrated in the present study. In fact, the production of formate and ethanol was not detected in the present study (data not shown). In the case of future industrial use of the LAC strain, this character will contribute to save the cost for the separation process of d-lactate. However, the titers and yields of d-lactate reported in other studies using glucose as a sole carbon source were greater than those obtained in the present study [21–23], indicating that further genetic modification of the PYR strain would facilitate increased productivity of various pyruvate derivatives. For example, the succinate biosynthetic pathway is still present in the PYR strain. Since succinate production is activated under anaerobic conditions to maintain redox balance [24,25], succinate might be produced as an unnecessary metabolite during the anaerobic production of pyruvate derivatives. Previous reports have demonstrated that the inactivation of frd suppresses the production of succinate under anaerobic conditions [21,26,27], suggesting that this strategy may lead to increased d-lactate production by derivatives of the LAC strain. Another route for improved productivity might be the inactivation of respiratory chain enzymes such as ATPase and cytochrome oxidases, which would lead to an increased rate of glucose uptake [28–30]. Since these modifications can be applied to the PYR strain, further improvements in the productivity of pyruvate derivatives can be expected.

5. Conclusions In this study, we constructed a new d-lactate-producing strain, LAC, by modifying the pyruvate-producing PYR strain. The LAC strain expresses the L. bulgaricus DLDH-encoding gene under the control of a T7-driven expression system. The d-lactate productivity of LAC strain was increased from 0.2 0.1 g L 1 to 12.1 1.6 g L 1 by limiting the aeration of the culture after 36 h, suggesting that ± · − ± · − preventing NADH consumption by O2-respiration was important. Furthermore, the cell density was increased by the pre-induction of the DLDH gene, resulting in an improvement in d-lactate productivity to 21.7 1.4 g L 1. The final concentration of d-lactate obtained in the present study (21.7 1.4 g L 1) ± · − ± · − was compatible with the pyruvate production by PYR strain (26.1 0.9 g L 1). These results suggested ± · − that we succeeded in the effective conversion of pyruvate to d-lactate in the LAC strain, demonstrating not only the versatility of the PYR strain but also the improvement of d-lactate productivity of the LAC strain when comparing with that of the old-LAC strain. Further engineered LAC strain may be used for applications such as industrial poly- production. Fermentation 2020, 6, 70 9 of 10

Supplementary Materials: The following are available online at http://www.mdpi.com/2311-5637/6/3/70/s1, Figure S1: Specific d-lactate production rates after 36 h by LAC strain under the air-regulated (red) and the combined (blue) conditions. Author Contributions: Conceptualization, K.W., T.F., H.A., T.M.; methodology, K.W., T.F., H.I., H.A.; validation; K.W.; formal analysis, K.W.; investigation, K.W.; resources, K.W.; data curation, K.W., H.I., T.M., T.F.; writing—original draft preparation, K.W.; writing—review and editing, K.W., T.F., H.I., H.A., T.M., A.M.; visualization, K.W.; supervision, T.F.; funding acquisition, K.W. All authors have read and agreed to the published version of the manuscript. Funding: This study was supported by Basic Research Funding of the National Institute of Advanced Industrial Science and Technology (AIST) and JSPS Grant Number 19K15372, Japan. Acknowledgments: We thank Dai Kitamoto and Yusuke Nakamichi for critical discussions; and Nobutaka Nakashima for technical support. Conflicts of Interest: The authors declare no conflict of interest.

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