Enzymatic Cascades for Efficient Biotransformation of Racemic

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Enzymatic Cascades for Efficient Biotransformation of Racemic Lactate Derived from Corn Steep Water Zhong Li,† Manman Zhang,† Tongtong Jiang,† Binbin Sheng,‡ Cuiqing Ma,† Ping Xu,§ and Chao Gao*,† †

State Key Laboratory of Microbial Technology, Shandong University, 27 Shanda South Road, Jinan 250100, PR China School of Environmental Science and Engineering, Sun Yat-sen University, Guangzhou 510275, PR China § School of Life Sciences & Biotechnology, Shanghai Jiao Tong University, 800 Dongchuan Road, Shanghai 200240, PR China ‡

S Supporting Information *

ABSTRACT: The corn starch industry produces a large amount of corn steep water, leading to high organic waste load. Lactate could be separated from corn steep water at a low cost, which supports the value-added utilization of corn steep water. However, the racemic characteristic of lactate from corn steep water restricts the development of this promising process. In this study, using D-lactate oxidase (D-LOX) from Gluconobacter oxydans, L-lactate oxidase (L-LOX) from Pediococcus sp., pyruvate decarboxylase from Zymomonas mobilis, and catalase from bovine liver, we synthesized an in vitro enzymatic system, including different enzymatic cascades, for the production of valuable platform chemicals from lactate separated from the corn steep water. Pyruvate was produced as an important intermediate and further converted into C2 (acetaldehyde) and C4 (acetoin) platform chemicals at high yields using optimized concentrations of pyruvate decarboxylase. The in vitro enzymatic system not only provides a novel technology platform for the production of optical lactate and lactate derivatives but also supports the sustainable development of corn starch industry. KEYWORDS: Lactate, Corn steep water, D-Lactate oxidase, L-Lactate oxidase, Pyruvate decarboxylase, Acetaldehyde, Acetoin

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processes.9 Advances in purification technologies have enabled the technologically feasible separation of lactate from corn steep water.10,11 A total of approximately 3000 tons of lactate is produced annually currently, and a production line with an annual output of 30 000 tons is under construction by Derunyuan Bio-Tech Co., Ltd. (Binzhou, China). The lactate content that can be practically separated from corn steep water in United States is about 528 125 tons (computation can be found in Supporting Information for annual production of lactate derived from corn wet milling), which is much higher than the annual world market demand in 2017 (367 300 tons).12 Currently, lactate is commercially manufactured through chemical synthesis and microbial fermentation. Microbial fermentation of starch produces approximately 90% of the total lactate around the world.13 Depending on the used strains, microbial fermentation is capable of efficiently producing optically pure D- or L-lactate under moderate conditions. However, the manufacturing cost of lactate fermentation remains rather high,12 although several efforts have been

INTRODUCTION The increasing world population is leading to an increasing demand for food supply, resulting in the generation of large amounts of polluting organic waste from the food industry. For example, 25 million tons of cornstarch is produced annually in the United States, accompanied by the production of about 16 million m3 of corn steep water (computational procedure for annual production of corn steep water during corn wet milling can be found in Supporting Information). The corn steep water is organic in nature and characterized by its high biological and chemical oxygen demand.1,2 Subjecting the corn steep water to evaporation produces corn steep liquor, a feedstock used in industrial production.3−5 However, this process suffers from high energy consumption and scale formation on tubes of the evaporators.6 Thus, it is desirable to find other economically viable corn steep water utilization processes to reduce the pollution in corn starch industry. One of the economically viable processes is separating important chemicals such as myo-inositol and proteins from corn steep water.7,8 A natural Lactobacillus-aided fermentation process, in which reducing sugars are converted to lactate, occurs during the corn steeping process. Lactate is one of the most abundant chemicals in the corn steep water with content of about 20−50 g/L, depending on the different steep © 2017 American Chemical Society

Received: January 13, 2017 Revised: February 21, 2017 Published: March 10, 2017 3456

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of pyruvate. The inhibition effects of pyruvate on D-LOX, D-lactate on L-LOX, and L-lactate on D-LOX were also assayed by the same method. Racemic Lactate Resolution and Pyruvate Production. To produce optically pure L-lactate and pyruvate from racemic lactate obtained from corn steep water, reactions were carried out in 20 mL reaction mixture containing 0.5 U/mL D-LOX, 0.1 mg/mL catalase, 20.7 mM racemic lactate, and 50.0 mM Tris-HCl buffer (pH 7.0). The reaction solutions were placed in 100 mL open conical flasks and incubated in a water bath shaken at 120 rpm and 30 °C. To produce optically pure D-lactate and pyruvate from racemic lactate, D-LOX was replaced by 0.05 U/mL L-LOX, while all other reaction conditions remained unchanged. Pyruvate production from racemic lactate was achieved using a combination of 0.5 U/mL D-LOX, 0.05 U/mL LLOX, and 0.1 mg/mL catalase. A sample (0.2 mL) was withdrawn at indicated time intervals, heated at 100 °C for 15 min to denature the proteins, centrifuged at 17 500 × g for 15 min, filtered, and subsequently analyzed using a high-performance liquid chromatography (HPLC) system. PDC Optimization for Directionally Controlled Pyruvate Transformation. The transformation of pyruvate with different concentrations of PDC (2.0, 5.0, 10.0, and 100.0 μg/mL and 1 and 2 mg/mL) was assayed at 30 °C in a 20 mL reaction mixture containing 50.0 mM Tris-HCl (pH 7.0), 20.0 mM pyruvate, 0.2 mM TPP, 2.0 mM MgSO4, and PDC. Samples were withdrawn at 0.5 h intervals to determine the concentrations of pyruvate, acetaldehyde, and acetoin. Racemic Lactate Resolution with Acetaldehyde/Acetoin Production. To produce optically pure L-lactate and acetaldehyde from racemic lactate obtained from corn steep water, 20 mL reaction solution containing 50.0 mM Tris-HCl (pH 7.0), about 20 mM racemic lactate, 0.5 U/mL D-LOX, 0.1 mg/mL catalase, 5.0 μg/mL PDC, 0.2 mM TPP, and 2.0 mM MgSO4 was placed in 100 mL open conical flasks, incubated in a water bath, and shaken at 120 rpm at 30 °C. To produce optical D-lactate and acetaldehyde, D-LOX was replaced by 0.05 U/mL L-LOX. Transformation of racemic lactate into acetaldehyde was achieved with the combination of D-LOX, L-LOX, catalase, and 5.0 μg/mL PDC. Samples were taken at 1 h intervals to assay the concentrations of lactate, pyruvate, acetaldehyde, and acetoin. To resolve racemic lactate with acetoin production, 2 mg/ mL PDC was used instead of 5.0 μg/mL PDC. Analytical Methods. The concentrations of lactate, pyruvate, and byproducts were determined using an HPLC system, as described by Li et al.17 Stereoselective assay of lactate was performed using an HPLC system (Agilent 1100 series, Hewlett-Packard) equipped with a chiral column (CRS10W, 46 mm × 50 mm) and a UV−vis detector working at 254 nm. The analysis was carried out at 25 °C with a mobile phase of 2.0 mM CuSO4 at a flow rate of 0.5 mL/min. Acetoin was analyzed using gas chromatography (GC), as described by Ma et al.18 The content of acetaldehyde was measured in 1 mL of phosphatebuffered saline (67.7 mM, pH 8.6) containing 0.2 mM NADH, the appropriate alcohol dehydrogenase from S. cerevisiae, and the reaction solution at 30 °C. The decrease in absorption at 340 nm was measured using a UV−visible spectrophotometer (Ultrospec 2100 pro; Amersham Biosciences). Two standard curves were obtained from a series of acetaldehyde solutions of known concentrations (0.25−5.0 mM and 5.0−20.0 mM). Lineweaver−Burk double-reciprocal plots were utilized to prepare the standard curves.

devoted to strain improvement and production engineering.14,15 Compared with lactate fermentation process using starch, lactate production from corn steep water may eliminate the need for starch. Additionally, this lactate production process could also promote the efficient reuse of corn steep water and reduce pollution from wet milling industry. However, contrary to the microbial fermentation of starch, where only one lactateproducing strain is used, numerous lactate-producing Lactobacillus strains participate in lactate production in the corn steeping process. Thus, only racemic lactate could be produced, followed by separation from corn steep water. The optical purity of lactate is a crucial factor in lactate-based industries. The key factor restricting the utilization of lactate from corn steep water is the outlet of the racemic lactate. In this study, different enzymatic cascades were exploited to convert the racemic lactate separated from corn steep water into C2 (acetaldehyde), C3 (pyruvate) and C4 (acetoin) platform chemicals. These cascades can also be used for the production of optically pure lactate (L- or D-lactate) by using selective biocatalysts.

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MATERIALS AND METHODS

Materials. Lactate separated from corn steep water was a kind gift from Derunyuan Bio-Tech Co., Ltd. (Binzhou, China). Reduced βnicotinamide adenine dinucleotide (NADH), isopropyl-β-D-1-thiogalactopyranoside (IPTG), thiamine pyrophosphate (TPP), alcohol dehydrogenase from Saccharomyces cerevisiae, catalase (CAT) from bovine liver, and L-lactate oxidase (L-LOX) from Pediococcus sp. were purchased from Sigma-Aldrich (St. Louis, MO, USA). All other chemicals were of analytical grade and commercially available. Preparation of Biocatalysts. D-Lactate oxidase (D-LOX) from Gluconobacter oxydans 621H (gene ID: 29627297) was purified as described by Sheng et al.16 To prepare pyruvate decarboxylase (PDC) from Zymomonas mobilis, the gene Zmpdc (codon-optimized for Escherichia coli expression, see Supporting Information) was synthesized by Sangon (Shanghai, China) and ligated into restriction sites of BamHI/SalI of plasmid pETDuet-1. The resulting recombinant plasmid pETDuet-Zmpdc was transferred into E. coli BL21(DE3) for protein expression. The recombinant E. coli BL21/pETDuet-Zmpdc was cultured in Luria−Bertani (LB) medium supplemented with 100.0 μg/mL ampicillin at 37 °C with shaking at 180 rpm to mid log phase (OD600nm = 0.4−0.8). It was cultivated for another 12 h at 20 °C with the addition of 1 mM IPTG to induce the expression of His-tagged PDC. Then, cells were harvested and washed twice in phosphate solution buffer (67.0 mM, pH 7.4) at 6000 rpm for 10 min. E. coli BL21/pETDuet-Zmpdc was resuspended in binding buffer (15.0 mM Na2HPO4·12H2O, 5.0 mM KH2PO4, 500.0 mM NaCl, and 20.0 mM imidazole, pH 7.4) supplemented with 10% v/v glycerol and 1.0 mM phenylmethylsulfonyl fluoride (PMSF), and lysed by sonication on ice. The remaining intact cells and cell debris were removed through centrifugation at 13 500 × g at 4 °C for 1 h. The supernatant was filtered through a 0.22 μm poly(ether sulfone) (PES) filter before being loaded onto a 5 mL HisTrap HP column (GE Healthcare, Sweden). A gradient ratio of elution buffer (15.0 mM Na2HPO4·12H2O, 5.0 mM KH2PO4, 500.0 mM NaCl, and 500.0 mM imidazole, pH 7.4) was applied to elute the target protein. The purified PDC was verified through SDS-PAGE, and the results are shown in Figure S1. Then, PDC was concentrated by ultrafiltration, desalted using a 5 mL HisTrap desalting column (GE Healthcare, Sweden), frozen in liquid nitrogen, and preserved at −80 °C until use. Determination of Enzymatic Characteristics. The kinetic parameters of LOXs were measured with a Clark-type oxygen electrode, as described by Sheng et al.16 Similarly, the inhibition effect of pyruvate on L-LOX was determined at 30 °C in 500 μL of airsaturated Tris-HCl buffer (50.0 mM, pH 7.0) containing 20.0 mM Llactate, an appropriate amount of L-LOX, and various concentrations

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RESULTS Design of Enzymatic Cascades for Racemic Lactate Utilization. An in vitro enzymatic system based on purified enzymes has become an innovative biomanufacturing platform because of its advantages over using living microorganisms. To exploit a technologically and economically feasible in vitro process based on racemic lactate, the fundamental principle is as follows: Minimize the number of enzymes required, and avoid exogenous addition of costly coenzymes.19,20 L-LOX, which is able to oxidize L-lactate and transfer electrons directly to O2 to form pyruvate and H2O2, undoubtedly satisfies the 3457

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Figure 1. Scheme of platform chemicals production from lactate separated from corn steep water through biotransformation.

Figure 2. Racemic lactate resolution and pyruvate production. (A) Scheme of lactate resolution and pyruvate production. (B) Time courses of production of optical L-lactate and pyruvate. (C) Time courses of production of optical D-lactate and pyruvate. (D) Time courses of pyruvate production from racemic lactate. (E) Inhibition effect of pyruvate on D-LOX. (F) Inhibition effect of pyruvate on L-LOX. Key: (black) pyruvate; (red) D-lactate; (blue) L-lactate. Results are means ± SD of three parallel experiments. 3458

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Figure 3. Racemic lactate resolution and acetaldehyde production. (A) Scheme of lactate resolution and acetaldehyde production. The PDC was used at a concentration of 5.0 μg/mL. (B) Time courses of production of optical L-lactate and acetaldehyde. (C) Time courses of production of optical D-lactate and acetaldehyde. (D) Time courses of acetaldehyde production from racemic lactate. Key: (black) pyruvate; (red) D-lactate; (blue) L-lactate; (pink) acetaldehyde; (teal) acetoin. Results are means ± SD of three parallel experiments.

demand for in vitro catalysis. Recently, we identified GOX2071 from G. oxydans as a D-LOX that could catalyze the oxidization of D-lactate with O2.16,21 This enzyme exhibits catalytic properties similar to those of L-LOX, except for the stereoselectivity. Thus, coproduction of pyruvate and optically pure lactate from racemic lactate could be realized by selectively using L-LOX or D-LOX (Figure 1). Pyruvate is an important C3 platform chemical, which could be easily transformed into other valuable molecules. The conversion of lactate into value-added chemicals via pyruvate should further expand the application scope of racemic lactate derived from corn steep water. Acetaldehyde is a representative of C2 platform chemicals, and acetoin is a representative of C4 platform chemicals. Both acetaldehyde and acetoin have broad industrial applications and are now principally manufactured from fossil resources. Acetaldehyde/acetoin could be produced from pyruvate through PDC catalyzing bond cleavage/ condensation reactions. Coupled with PDC, different enzymatic cascades based on LOX(s) could be developed for acetaldehyde/acetoin production, accompanied by resolution of racemic lactate (Figure 1). Racemic Lactate Resolution for Production of Pyruvate and Optically Pure Lactate. The lactate separated from corn steep water by Derunyuan Bio-Tech Co., Ltd. (Binzhou, China) contained 41.6% L-lactate and 58.4% Dlactate (Figure S2). To extend the scope of application of this raw material, D-LOX was employed to biotransform racemic lactate into L-lactate and pyruvate (Figure 2A). H2O2 generated

during the oxidation of D-lactate was decomposed by catalase from bovine liver to avoid the spontaneous oxidative cleavage of pyruvate. As shown in Figure 2B, racemic lactate (20.7 mM) was optically resolved by 0.5 U/mL D-LOX in 7 h. D-Lactate (12.1 mM) was transformed into 11.7 mM pyruvate with a yield of 96.7%, while 8.5 mM L-lactate with an ee value >99% (Figure S2E) was detected at a residual rate of 98.2%. It is notable that the transformation of D-lactate decreased with the accumulation of pyruvate. This decrease in the transformation rate might be attributed to feedback inhibition of pyruvate to D-LOX (Figure 2E), while the inhibition of L-lactate is negligible when the concentration is below 10.0 mM (Figure S3A). Consequently, it would be desirable to increase the transformation rate by further conversion of pyruvate into other value-added chemicals. To transform racemic lactate into D-lactate and pyruvate, commercially available L-LOX from Pediococcus sp. (SigmaAldrich) was combined with catalase from bovine liver. The kinetic parameters of L-LOX were measured using an oxygen electrode (Table S1). The Km and Vmax values toward L-lactate were estimated to be 0.52 mM and 90.1 U/mg, respectively. The Km of L-LOX toward O2 was determined to be 0.19 mM, which was similar to that of D-LOX, while the Vmax toward O2 was 213.9 U/mg. As shown in Figure 2C, 8.6 mM pyruvate was produced from L-lactate by 0.05 U/mL L-LOX in 7 h with a yield of 99.2%, while 12.0 mM D-lactate was detected at a residual rate of 99.7% with an ee value >99% (Figure S2F). 3459

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Figure 4. Racemic lactate resolution and acetoin production. (A) Scheme of lactate resolution and acetoin production. The PDC was used at a concentration of 2 mg/mL. (B) Time courses of production of optical L-lactate and acetoin. (C) Time courses of production of optical D-lactate and acetoin. (D) Time courses of acetoin production from racemic lactate. Key: (red) D-lactate; (blue) L-lactate; (pink) acetaldehyde; (teal) acetoin. Results are means ± SD of three parallel experiments.

and 10.0 μg/mL totally consumed pyruvate in 1 h. A further increase in PDC concentration (exceeding 100.0 μg/mL) resulted in less than 0.5 h being required for pyruvate consumption. As expected, the concentration of acetaldehyde decreased, while the concentration of acetoin increased with the increase in PDC concentration (Figure S5C,D). The maximal temporal accumulation of acetaldehyde was observed at 1 h with 5.0 μg/mL PDC. Thus, 5.0 μg/mL PDC could be coupled with LOX(s) for acetaldehyde production from lactate. At 3.5 h, 8.8 mM acetoin accumulated from 20.0 mM pyruvate at a yield of 88.2% with 2 mg/mL PDC. Accordingly, 2 mg/mL PDC was coupled with LOX(s) to produce acetoin with high yield. Racemic Lactate Resolution with Acetaldehyde Production. To explore a sustainable route for acetaldehyde production from racemic lactate derived from corn steep water, L -LOX/ D-LOX and PDC were employed (Figure 3A). Production of optically pure L-lactate and acetaldehyde was achieved using a combination of 0.5 U/mL D-LOX and 5.0 μg/ mL PDC. As shown in Figure 3B, 8.3 mM L-lactate with an ee value >99% (Figure S2G) was recovered from 20.3 mM racemic lactate at a residual rate of 97.7%. Acetaldehyde accumulated to a maximum of 8.8 mM at 4 h and decreased thereafter. The yield of acetaldehyde was calculated to be 73.8%. No pyruvate or D-lactate was detected in the final reaction solution. As shown in Figure 3C, through the combination of 0.05 U/mL L-LOX with 5 μg/mL PDC, 11.5 mM D-lactate with an ee value >99% (Figure S2H) was detected at 5 h with a residual rate of 97.3%. Acetaldehyde

The inhibition effect of pyruvate and D-lactate on L-LOX was also analyzed (Figures 2F and S3B). Nearly 50% loss of L-LOX activity was caused by 5.0 mM pyruvate or 100.0 mM D-lactate. Considering that L-LOX was inhibited less strongly than D-LOX by pyruvate, the ratio of L-LOX content to D-LOX content (1:1, 1:5, 1:10, 1:15, and 1:20) was optimized to synchronize the reaction time for pyruvate production from racemic lactate by L-LOX and D-LOX (Figured S4 and 2D). Transformation of racemic lactate into pyruvate was carried out with the combination of L-LOX and D-LOX at an optimized ratio of 1:10. As shown in Figure 2D, 20.3 mM pyruvate was produced from 20.7 mM racemic lactate in 7 h at a yield of 97.9%. Directionally Controlled Biotransformation of Pyruvate by Adjusting PDC. Pyruvate is the substrate of PDC, the first enzyme in ethanol fermentation. In addition to catalyzing the decarboxylation of pyruvate into acetaldehyde, PDCs from various species are capable of transferring hydroxyethyl group to another acetaldehyde, forming acetoin (Figure S5A).22−24 Considering that the carboligation process catalyzed by PDC is a further reaction based on the decarboxylation process, the composition of reaction products might be tightly related to the concentration of this enzyme. To efficiently produce acetaldehyde or acetoin from lactate, the concentration of PDC was optimized through the incubation of 20.0 mM pyruvate and PDC from Z. mobilis at different concentrations. The consumption of pyruvate and accumulation of acetaldehyde and acetoin were investigated. As shown in Figure S5B, PDC (2.0 μg/mL) required 3 h to completely consume 20.0 mM pyruvate. PDC concentrations between 5.0 3460

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used in enzymatic systems. Previously, Sheng et al. screened several candidate D-iLDHs for soluble D-LOXs. Among them, D-LOX in G. oxydans showed high catalytic activity and stereoselectivity toward D-lactate using O2 as a direct electron acceptor.21 However, L-LOXs from various species were able to meet the specific catalytic requirements.29−31 The commercialized L-LOX from Pediococcus sp. exhibited good solubility, high catalytic activity, and stereoselectivity toward L-lactate. Consequently, the two selective biocatalysts, L-LOX from Pediococcus sp. and D-LOX from G. oxydans, have been used in the production of L-lactate, D-lactate, and pyruvate from racemic lactate in this study. The commercialized and frequently used catalase from bovine liver was utilized to remove H2O2 produced during the oxidization of lactate. Using the in vitro synthesized enzymatic cascades, optically pure lactates and pyruvate were produced with high ee and high yield. However, since pyruvate exhibited an apparent inhibitory effect on both D-LOX and L-LOX, the enzymatic system introduced in this study should not be feasible to produce pyruvate in large scale. However, since pyruvate is a key central metabolite, the conversion of lactate into pyruvate should be the first step for further transformation into value-added chemicals. The conversion of racemic lactate into pyruvate is the foundation for further utilization of racemic lactate separated from corn steep water. Acetaldehyde, which is mainly obtained by the direct catalytic oxidation of ethylene by the Wacker process, is an important intermediate in organic synthesis.32 Biotechnological production of acetaldehyde through fermentation processes suffered because of its high toxicity toward microorganisms. Therefore, combination of PDC with the synthesized pyruvate production cascades would eliminate the cell-associated barriers and remit the inhibition effect of pyruvate on LOXs. PDC in Z. mobilis has been intensively studied with respect to the catalytic mechanism and carboligase reactions.24 Moreover, it exhibited high stability and a half-life of 22 h at 50 °C and higher than 100 h at room temperature.19,24 In this study, using the enzymatic system containing LOX(s) and PDC from Z. mobilis (5.0 μg/mL) under mild reaction conditions, 13.6 mM acetaldehyde was produced with a yield of 66.9%. Many chemical catalysts have also been explored for the production of acetaldehyde from lactate.33−38 These inorganic catalytic reactions suffer from the difficult preparation of catalysts, harsh reaction conditions, and influence of end gas on the purity of acetaldehyde. For example, a photocatalytic route for the conversion of lactate to acetaldehyde in water was demonstrated recently. The yield of acetaldehyde was 39%, which was lower than of the biocatalytic process utilizing LOX(s) and PDC.39 Although microbiological synthesis of acetoin has been exhaustively studied, condensation of acetaldehyde into acetoin could be an alternative way of producing this C4 platform chemical. Recently, several chemical methods for the acetoin production from acetaldehyde have been disclosed.40,41 Besides decarboxylase activity, PDCs from several species have been verified to possess acetoin producing carboligation activity from pyruvate.22−24 In this work, we found that the yield of acetaldehyde decreased with elevated PDC concentration, while the yield of acetoin from pyruvate was positively related with the concentration of PDC. Using the enzymatic system containing LOX(s) and PDC from Z. mobilis (2 mg/mL) under mild reaction conditions, 9.8 mM acetoin was produced from 21.1 mM racemic lactate with a high yield of 92.7%. This

accumulated to a maximum of 6.3 mM with a yield of 74.8%. Production of acetaldehyde from racemic lactate was also achieved from combined assembly of D-LOX, L-LOX, and 5.0 μg/mL PDC. As shown in Figure 3D, the temporary maximum concentration of acetaldehyde was 13.6 mM at 4 h with a calculated yield of 66.9%. Concentrations of intermediate metabolites and byproducts were also determined. No accumulation of hydrogen peroxide was detected. Pyruvate was totally transformed at 5 h in all the reaction systems (Figure 3). A small amount of acetoin (less than 0.34 mM) accumulated during acetaldehyde production, possibly as a result of further reaction of acetaldehyde. Racemic Lactate Resolution with Acetoin Production. According to the optimization results, 2 mg/mL PDC coupled with LOX(s) was used for resolution of racemic lactate with acetoin production (Figure 4A). As shown in Figure 4B, 12.2 mM D-lactate was consumed to produce 5.4 mM acetoin at a yield of 88.4% in 5 h. L-Lactate (8.9 mM) remained at a rate of 99.7%, and the ee value was >99% (Figure S2I). As shown in Figure 4C, 3.9 mM acetoin was produced from 8.9 mM Llactate at a yield of 86.7%. D-Lactate at a concentration of 12.2 mM was detected at a residual rate of 99.9% and an ee value >99% (Figure S2J). Acetoin production was achieved using a combination of D-LOX, L-LOX, and 2 mg/mL PDC. As shown in Figure 4D, 9.8 mM acetoin was produced from 21.1 mM racemic lactate at a yield of 92.7%. During the whole catalytic processes, no pyruvate was detected, which might be caused by the high concentration of PDC. The concentration of acetaldehyde in these reaction systems was also determined. The content of acetaldehyde increased in the first hour, kept constant in the next 3 h and decreased thereafter (Figure 4B− D). The decrease of acetaldehyde after 4 h might result from the biotransformation of acetaldehyde into acetoin by PDC.

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DISCUSSION Manufacturing corn starch through the wet milling process has been put into effect since 170 years ago. During the steeping process, fermentation of soluble components is conducted mainly by numerous Lactobacillus strains. Thus, racemic lactate is produced in high concentrations, and it improves the yield of corn starch and increases the solubility of protein.25,26 Development of purification technologies has made separation of lactate from corn steep water relatively easy. However, the application scope of racemic lactate separated from corn steep water is limited because of the requirement of high optical purity. Herein, we explored different enzymatic cascades for comprehensive utilization of this abundantly available feedstock. L-Lactate, D-lactate, and pyruvate are three C3 platform chemicals that can be produced from racemic lactate separated from corn steep water by biotransformation. The key step for production of these three chemicals is the selective dehydrogenation of either or both isomers of lactate to pyruvate. Both NAD-dependent lactate dehydrogenases (nLDHs) and NAD-independent lactate dehydrogenases (iLDHs) might be employed in the enzymatic synthesis of pyruvate from lactate.15 Compared with nLDHs that require NAD as a cofactor, iLDHs could catalyze lactate oxidation by avoiding expensive NAD addition and cofactor regeneration processes.27 However, most of these enzymes are membranebound and can only be used in whole cell biocatalysis.28 It is desirable to explore biocatalysts that have good solubility, are independent of expensive cofactors, and are capable of being 3461

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ACS Sustainable Chemistry & Engineering is the first report of acetoin production from lactate, and has the potential to further extend application of lactate produced from biomass. The in vitro enzymatic system is now transitioning from being a fundamental research tool to an applicable biomanufacturing platform. Numerous enzymatic systems based on glucose, xylose, sucrose, and glycerol have been reported.19,20,42−47 Compared with the in vivo biosystems, the in vitro synthetic systems are superior in several aspects. First, the elimination of transmembrane transport process might result in faster reaction rates. Second, the reduced metabolic complexity avoids the production of undesired products and improves the yield of aimed chemicals. For instance, pyruvate, which acted as an important intermediate in this work, is a central metabolite of microorganisms and is readily converted into various chemicals within cells. Third, exclusive use of purified enzymes would improve the tolerance of toxic compounds of the catalytic systems.19,46 For instance, the toxic effect of acetaldehyde to the cell is one of the main obstacles limiting the bioproduction of acetaldehyde. The enzymatic cascades introduced in this study might provide an alternative for the sustainable production of acetaldehyde. Fourth, all the reaction conditions of enzymatic systems are under control, guaranteeing the catalysis of unnatural reactions.20,48 However, readily available enzymes (e.g., LLOXs) need to be further explored for lowering the cost of the enzyme preparation and extending application of the enzymatic system. Moreover, due to low thermostability of D-LOX and LLOX, this enzymatic system could not be used at high temperatures. Further work targeting development of thermostable enzymes might improve the applicability of the enzymatic system. In addition to the elimination of unwanted metabolic pathways, the in vitro enzymatic systems also have advantages in product purification process.20,45,46 In this study, the separation of products from the biocatalytic systems would be easy to perform with only one target product, for instance, pyruvate from the system containing D-LOX and L-LOX, acetaldehyde from the system containing D-LOX, L-LOX, and 5.0 μg/mL PDC, or acetoin from the system containing DLOX, L-LOX, and 2 mg/mL PDC. Since the boiling points of acetaldehyde, lactate, and acetoin are 20.8, 122, and 148 °C, respectively, fractional distillation with low cost could be used in separation of products from the reaction systems with two target products (optical lactate and acetaldehyde/optical lactate and acetoin). Different potentially useful chemicals, including pyruvate, acrylic acid, 1,2-propanediol, and lactate ester have been produced from lactate through chemical or biotechnological routes.10,15,49,50 Thus, lactate is regarded as a feedstock for green chemistry of the future. In this study, the application of lactate was further extended through biotransformation of lactate into two important platform chemicals: acetaldehyde and acetoin. Optical lactate fermentation is now operated commercially using sugars present in the biomass. Unfortunately, optical lactate fermentation industry currently suffers from high substrate cost and operating cost.12 Corn steep water is a byproduct and major pollutant of the corn wet milling industry with a very high waste load. Numerous chemicals, such as inositol, proteins, amino acids, and racemic lactate, could be separated from corn steep water.7,8,51,52 Using the enzymatic cascades introduced in this work, optical lactate could be produced and easily separated from the enzymatic system. Our

work not only introduced a promising strategy supporting the resource reuse of corn steep water but also proposed a novel alternative for the optical lactate production. The rapidly growing world population requires an increased food supply, which is one of the main causes of environmental pollution throughout the world. Corn wet milling is a significant industrial activity, but it generates a high waste load of corn steep water. In this work, less than four enzymes were combined into an in vitro enzymatic system producing optically pure lactates, acetaldehyde, and acetoin from racemic lactate separated from corn steep water with high yields. More value-added platform chemicals might also be produced from racemic lactate through further development of other biotransformation systems. Thus, separation and utilization of racemic lactate from corn steep water have potential in providing low-cost lactate and supporting the sustainable development of wet milling industry and deserve further indepth investigation.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acssuschemeng.7b00136. Computational procedure for annual production of lactate derived from corn wet milling, additional table and figures, and codon-optimized nucleic acid sequence of gene Zmpdc (PDF)

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Tel.: 86-531-88369463. Fax: 86531-88369463. ORCID

Zhong Li: 0000-0003-2389-1443 Tongtong Jiang: 0000-0002-0537-9473 Ping Xu: 0000-0002-4418-9680 Chao Gao: 0000-0002-5205-0670 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This study was supported by the National Natural Science Foundation of China (31470164 and 31270856), the Chinese National Program for High Technology Research and Development (2014AA021206), and the Young Scholars Program of Shandong University (2015WLJH25).

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