AEM Accepted Manuscript Posted Online 10 April 2015 Appl. Environ. Microbiol. doi:10.1128/AEM.00527-15 Copyright © 2015, American Society for Microbiology. All Rights Reserved.

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Running title: D-Lactate Utilization of Gluconobacter oxydans

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Utilization of D-Lactate as an Energy Source Supports the Growth of

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Gluconobacter oxydans

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Binbin Sheng,a Jing Xu,a Yingxin Zhang,a Tianyi Jiang,a Sisi Deng,a Jian Kong,a Chao

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Gao,a,# Cuiqing Ma,a and Ping Xub

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State Key Laboratory of Microbial Technology, Shandong University, Jinan 250100,

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People’s Republic of Chinaa;

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State State Key Laboratory of Microbial Metabolism and School of Life Sciences and

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Biotechnology, Shanghai Jiao Tong University, Shanghai 200240, People’s Republic of

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Chinab

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#

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Phone: +86-531-88364003; Fax: +86-531-88369463;

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Mailing address: State Key Laboratory of Microbial Technology, Shandong University,

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Jinan 250100, People’s Republic of China.

Corresponding author: Chao Gao; E-mail: [email protected];

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ABSTRACT

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D-Lactate

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growth of Gluconobacter oxydans 621H in this study. Interestingly, this strain used

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D-lactate

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lactate-utilizing bacteria. The enzymatic basis for the growth of G. oxydans 621H on

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D-lactate

was therefore investigated. Although two putative NAD-independent

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D-lactate

dehydrogenases, GOX1253 and GOX2071, were capable of oxidizing

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D-lactate,

GOX1253 was the only enzyme able to support the D-lactate-driven

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growth of the strain. GOX1253 was characterized as a membrane-bound

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dehydrogenase with a high activity towards D-lactate, while GOX2071 was

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characterized as a soluble oxidase with broad substrate specificity towards

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D-2-hydroxy

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feature that has not been reported previously in D-lactate oxidizing enzymes. This

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study not only clarifies the mechanism for the growth of G. oxydans on D-lactate, but

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also provides new insights for applications of the important industrial microbe and

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the novel D-lactate oxidase.

was identified as one of a few available organic acids that supported the

as an energy source but not as a carbon source, unlike other

acids. The latter used molecular oxygen as a direct electron acceptor, a

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2

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INTRODUCTION

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Gluconobacter oxydans is a Gram-negative, rod-shaped, obligate aerobic bacterium. The

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genomic information of G. oxydans 621H suggests that this bacterium harbors

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functionally impaired Embden-Meyerhof-Parnas (EMP) pathway and tricarboxylic acid

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(TCA) cycle (1-2). The respiratory chain of G. oxydans is different from other aerobic

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bacteria; it does not rely on NADH as an electron donor (1). Thus, the major function of

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the EMP pathway and TCA cycle in G. oxydans is to provide biosynthetic precursors (3).

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The energy supply in G. oxydans favors a mode of action, in which numerous

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dehydrogenases oxidize their substrates incompletely under growth conditions (2). High

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oxidation rates, correlating with low growth yields, give G. oxydans a unique capability of

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enabling the rapid and incomplete oxidation of a wide range of sugars and alcohols and

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make the strain a promising biocatalyst in a variety of biotechnological processes (3-7).

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However, relatively little is known about the membrane-bound and soluble

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dehydrogenases that catalyze the oxidation of specific substrates.

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In nature, G. oxydans mainly grows in sugary, acidic, and alcoholic niches including

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flowers, fruits, garden soil, cider, wine, and beer (3-4). The habitats of G. oxydans contain

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numerous organic compounds, such as sugars, alcohols, amino acids, and organic acids.

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Utilization of these organic compounds is vital for the survival of G. oxydans. The growth

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of G. oxydans 621H on various sugars and alcohols has been investigated in detail (8).

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Sugars and alcohols in the habitats of G. oxydans are usually oxidized rapidly, providing

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electrons for the generation of energy. The oxidation products, such as sugar acids, are

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difficult for other organisms to assimilate; when released into the environment, they

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inhibit the growth of competitors by lowering the pH (2). However, it is not known

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whether G. oxydans can use amino acids and organic acids, the two other types of 3

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compounds extensively distributed in nature, to support its growth. Many bacteria, such as Escherichia coli (9-10), Corynebacterium glutamicum (11),

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and Pseudomonas stutzeri (12), are able to utilize lactate for growth. During the lactate

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utilization process in these bacteria, lactate is first oxidized to pyruvate; pyruvate is then

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used as both carbon and energy sources to support the growth of these bacteria. Lactate

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can be oxidized to pyruvate by NAD-dependent lactate dehydrogenases (nLDHs) and

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NAD-independent lactate dehydrogenases (iLDHs) (13). The nLDHs often play

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important roles in anaerobic fermentation to produce lactate from pyruvate (14-15). The

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iLDHs usually serve as the key enzymes to support the growth of microorganisms on

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lactate (9, 11-13). Furthermore, the iLDHs can be divided into D-iLDHs and L-iLDHs

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according to their chiral specificity. Most characterized iLDHs are membrane-bound

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proteins that use quinones or cytochrome c as native electron acceptors (13). Two

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untypical L-iLDHs, L-lactate oxidase and L-lactate monooxygenase, use O2 directly as an

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electron acceptor (13). Based on this feature, these two L-iLDHs are widely used in

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biosensor and biocatalysis applications (16-18). However, no D-iLDH that directly uses

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O2 as an electron acceptor has been reported (13).

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In this study, we found that G. oxydans 621H is unable to utilize almost all amino

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acids and most common organic acids for growth. Only several 2-hydroxy acids,

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particularly D-lactate, supported the growth of G. oxydans 621H. However, pyruvate, the

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product of D-lactate oxidation, did not support the growth of G. oxydans in our

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observations. Thus, the driving force of D-lactate on G. oxydans 621H growth should

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derive solely from the process of D-lactate oxidation. To understand the utilization of

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D-lactate

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and identified two putative D-iLDHs (GOX1253 and GOX2071). The two putative

by G. oxydans 621H, we analyzed its genome, which was sequenced in 2005 (1),

4

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D-iLDHs

were characterized and their physiological functions were investigated to clarify

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the unusual mechanism of G. oxydans growth on D-lactate. Interestingly, we also found

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that GOX2071 is a novel type of D-iLDH that uses O2 as an electron acceptor, with which

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GOX2071 may be useful in biosensor and biocatalysis applications.

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MATERIALS AND METHODS

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Bacterial strains and culture conditions. The bacterial strains and plasmids used in this

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study are listed in Table 1. G. oxydans 621H (DSM 2343) and its derivatives were

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cultivated routinely in Y-S medium containing 80 g sorbitol, 24 g yeast extract, 5 g

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(NH4)2SO4, 2 g KH2PO4, and 5 g MgSO4·7H2O in 1 l of distilled water at 200 rpm and

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30°C (7, 19). Before G. oxydans 621H was inoculated to other mediums, the cells were

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washed with 0.85 % NaCl solution to remove the residual Y-S medium thoroughly. In the

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assays to compare the growth of G. oxydans 621H on different organic compounds, G.

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oxydans 621H was cultured in the mediums (pH 6.0) containing 5 g/l yeast extract and 3

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g/l peptone (abbreviated as YE-P medium) supplemented with 50 mM of D-glucose,

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1,2-propanediol, 19 proteinaceous amino acids except for tyrosine, or 24 organic acids,

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respectively. Tyrosine was not assayed as it would be precipitated at 50 mM once below

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pH 10. In the experiment of determination of the growth curve, G. oxydans 621H and its

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derivatives were cultivated in 500-ml shaking flasks filled with 50 ml liquid YE-P

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medium supplemented with 50 mM of D-lactate or pyruvate, respectively. E. coli strains

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were cultured in Luria–Bertani (LB) medium supplemented with appropriate antibiotics

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at 180 rpm and 37°C. Antibiotics were added, if necessary, at the following

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concentrations unless otherwise stated: cefoxitin at 50 μg/ml, kanamycin at 50 μg/ml, and

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ampicillin at 100 μg/ml. 5

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Deletion and complementation of GOX1253 and GOX2071 encoding genes. To

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delete the gene of GOX1253 in G. oxydans 621H, the homologous arms of upstream and

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downstream of the target GOX1253 gene were PCR amplified by using the primers

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GOX1253up.f/GOX1253up.r and GOX1253down.f/GOX1253down.r (Table 2). Then,

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they were fused together via recombinant PCR by using the primers GOX1253up.f and

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GOX1253down.r, which contained EcoRI and HindIII restriction enzyme sites,

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respectively (Table 2). The generated fusion construct and pK18mobsacB (20), a

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mobilizable plasmid that does not replicate in G. oxydans, were digested with EcoRI and

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HindIII, respectively, and were then linked by using T4 DNA ligase to form

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pK18mobsacB-ΔGOX1253. The plasmid was transferred into G. oxydans via a triparental

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mating method as described previously (21). E. coli DH5α harbouring

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pK18mobsacB-ΔGOX1253 was used as the donor strain, E. coli HB101 harbouring the

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plasmid pRK2013 as the helper strain, and G. oxydans 621H as the recipient strain. The

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Y-S medium agar plate supplemented with 50 μg/ml kanamycin, 50 μg/ml cefoxitin, and

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0.1% acetic acid (v/v) was used for screening single crossover mutant, in which acetic

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acid was used to eliminate E. coli growth (22). The mutants lost GOX1253 gene in a

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second homologous recombination were screened from the Y-S medium agar plate

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containing 10% sucrose. The ΔGOX1253 mutant was checked by PCR and sequenced by

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using primers GOX1253up.f and GOX1253down.r. Thereafter, the mutants, G. oxydans

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ΔGOX2071 and G. oxydans ΔGOX1253 ΔGOX2071, were constructed with the same

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method by using the primers GOX2071up.f/GOX2071up.r and

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GOX2071down.f/GOX2071down.r (Table 2).

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For the complementation of GOX1253 and GOX2071, fragments GOX1253 and GOX2071 were amplified by PCR using G. oxydans 621H genome as template and using 6

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GOX1253up.f/GOX1253down.r and GOX2071up.f/GOX2071down.r as primers,

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respectively (Table 2). The resulting PCR products were cloned into EcoRI/HindIII-cut

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pBBR1p264, a broad-host-range plasmid with a strong promoter derived from

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pBBR1MCS2 and used for the expression of proteins in G. oxydans (23). The resulting

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plasmids, pBBR1p264-GOX1253 and pBBR1p264-GOX2071, were conjugated into G.

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oxydans ΔGOX1253 ΔGOX2071 via the triparental mating method mentioned above.

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Purification of recombinant GOX1253 and GOX2071. The genes encoding

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GOX1253 and GOX2071 were amplified by PCR using genome of G. oxydans 621H as

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template and GOX1253C.f/GOX1253C.r and GOX2071C.f/GOX2071C.r as primers,

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respectively (Table 2). The PCR product of GOX1253 and pET25b were digested by

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NdeI and HindIII and linked by T4 DNA ligase to construct plasmid pET25b-GOX1253.

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The PCR product of GOX2071 was digested by NdeI and XhoI and cloned into

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NdeI-XhoI sites of pET25b to form plasmid pET25b-GOX2071. The resulting plasmids

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were separately transformed into E. coli C43 (DE3) (24) for protein expression. The

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recombinant E. coli strains were cultured in LB medium containing 100 μg/ml ampicillin

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at 37°C and 180 rpm to optical density of 0.4 ~ 0.6 at 600 nm, then 1 mM

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isopropyl-β-D-1-thiogalactopyranoside (IPTG) was added to induce protein expression.

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Cultures of E. coli C43 (pET25b-GOX1253) were grown at 16°C for a further 10 h, while

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E. coli C43 (pET25b-GOX2071) were further incubated at 37°C for 6 h. Thereafter, cells

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were harvested and washed twice in 67 mM phosphate buffer (pH 7.4) at 3,400 × g for 10

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min at 4°C.

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E. coli C43 (pET25b-GOX1253) cells were suspended in binding buffer (pH 7.4, 20

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mM sodium phosphate and 500 mM sodium chloride) supplemented with 0.1% Triton

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X-100, 1 mM phenylmethylsulfonyl fluoride (PMSF), and 10% glycerol, and disrupted 7

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by sonication with a sonics sonicator (500 W/20 KHz, USA) on ice bath. Intact cells and

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cell debris were removed by centrifugation at 13,500 × g and 4°C. The resultant

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supernatant was applied to a HisTrap HP column (5 ml) equilibrated with binding buffer

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supplemented with 0.1% Triton X-100, and eluted with a gradient ratio of elution buffer

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(pH 7.4, 20 mM sodium phosphate, 500 mM imidazole, and 500 mM sodium chloride)

163

supplemented with 0.1% Triton X-100 to obtain purified GOX1253.

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E. coli C43 (pET25b-GOX2071) cells were suspended in buffer A (pH 7.4, 20 mM

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sodium phosphate) supplemented with 1 mM PMSF, and 10% glycerol, disrupted by

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sonication, and centrifuged to remove intact cells and cell debris. The resultant

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supernatant was first loaded onto a Source 30Q column (2.5 × 25 cm) equilibrated with

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buffer A and eluted with a gradient ratio of buffer B (pH 7.4, 20 mM sodium phosphate,

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500 mM sodium chloride). The fractions containing D-iLDH activity were collected and

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then applied to a HisTrap HP column (5 ml). Thereafter, GOX2071 was purified with

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gradient elution by using elution buffer.

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The purified enzymes were concentrated by ultrafiltration. Finally, GOX1253 was

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stored in 50 mM Tris-HCl buffer (pH 7.4) supplemented with 0.1% Triton X-100, and

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GOX2071 was stored in 50 mM Tris-HCl buffer (pH 7.4).

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Ultracentrifugation for iLDHs localization. After cultured for 20 h, cells of G.

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oxydans 621H and its derivatives were harvested, washed twice with 67 mM phosphate

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buffer solution (pH 7.4), and resuspended in 20 mM phosphate buffer solution (pH 7.4)

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containing 1 mM PMSF. Then the suspension of the resulting cells was disrupted by a

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pressure cell homogenizer (Stansted Fluid Power LTD, UK) at a pressure of 1.5 bars.

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Intact cells were removed by conventional centrifugation at 13,500 × g and 4°C for 5 min.

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Thereafter, the resultant crude extract of 4 ml was ultra-centrifuged at 150,000 × g and 8

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4°C for 2 h. The resulting precipitate was completely separated from the

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ultracentrifugation supernatant, and subsequently suspended in the buffer of 4 ml, and

184

then subjected to successive enzyme determination.

185

Determination of iLDH activity using MTT as an electron acceptor. The activity

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of iLDH was assayed at 30°C in 0.8 ml of 50 mM Tris-HCl buffer (pH 7.4) containing 10

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mM D-lactate or L-lactate, 0.2 mM MTT

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[3-(4,5-dimethylthia-zol-2-yl)-2,5-diphenyltetrazolium bromide], 0.2 mM phenazine

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methosulfate (PMS), and appropriate crude extracts or enzyme solutions, unless

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otherwise stated. To determine the substrate specificities of enzymes, 10 mM sodium salts

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of different 2-hydroxy acids were used instead of lactate. The rate of MTT reduction was

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determined by measuring the absorbance change at 578 nm. One unit of iLDH activity

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was defined as the amount that catalyzed the reduction of 1 µmol of MTT per minute.

194

The protein concentration was determined by the Lowry procedure by using bovine

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serum albumin as the standard (25).

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Detection of D-lactate oxidase activity and its kinetics. D-Lactate oxidase activity

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was measured at 30°C in 500 μl reaction solution, which contained 50 mM Tris-HCl (pH

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7.4), 20 mM D-lactate, and enzyme, using a Clark-type oxygen electrode (Oxytherm,

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Hansatech, UK) equipped with automatic temperature controlled electrode chamber at

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900 rpm. One unit of oxidase activity was defined as the amount that catalyzed the

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reduction of 1 µmol of O2 per minute.

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To determine the apparent Km for O2, oxygen consumption was measured in 50 mM

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Tris-HCl buffer (pH 7.4) containing various concentrations of O2, appropriate enzyme,

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and 20 mM D-lactate, using a Clark-type oxygen electrode. The initial O2 levels in

205

reaction solution were varied by mixture of O2-saturated and N2-saturated Tris-HCl 9

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buffers (pH 7.4). When measuring the apparent Km for D-lactate, air-saturated Tris-HCl

207

buffer was used. Lineweaver-Burk double-reciprocal plots were utilized to determine the

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apparent kinetic parameters.

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Analytical procedures. The sodium dodecyl sulfate-polyacrylamide gel

210

electrophoresis (SDS-PAGE) was performed as reported previously (26). For

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size-exclusion chromatography analysis, protein samples were loaded onto a Superdex

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200 10/300 GL column (GE Healthcare) equilibrated with a buffer C (pH 7.2, 50 mM

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phosphoric acid, and 0.15 M NaCl) at a flow rate of 0.5 ml/min. Standard proteins,

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thyroglobulin (669 kDa), ferritin (440 kDa), aldolase (158 kDa), conalbumin (75 kDa),

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ovalbumin (43 kDa), and ribonuclease A (13.7 kDa), were used for standard curve.

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To determine the flavin cofactor of the purified proteins, standard flavins including

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FAD, FMN, and riboflavin were measured with high performance liquid chromatography

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(HPLC) (Agilent 1100 series) equipped with an Agilent Zorbax Eclipse XDB-C18 column

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(150 × 4.6 mm, 5 µm) and a variable-wavelength detector at 450 nm (27). The mobile

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phase consisted of 50 mM (NH4)2CO3 and methanol with a ratio of 82:18 (v/v) at a flow

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rate of 0.5 ml/min at 30°C (27). The purified proteins were heated at 100°C for 10 min,

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and then centrifuged at 16,000 × g for 15 min to remove denatured proteins. The resultant

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supernatants containing the cofactors released from the purified enzymes were analyzed

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with HPLC to identify the type of the cofactor. Standard FAD solutions of 1, 2, 5, 10, 20,

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40, 80, 120 µM were detected at 450 nm, and used for quantitative analysis.

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Lactate, pyruvate, and acetate were measured using HPLC equipped with an Aminex

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HPX-87H column (300 mm × 7.8 mm, 9 µm) and a differential refractive index detector

228

(RID) (28). The mobile phase was 10 mM H2SO4 at a flow rate of 0.4 ml/min at 55°C.

229 10

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RESULTS

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D-Lactate

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oxydans can thrive by using sugars and polyols, such as glucose and sorbitol, as the sole

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carbon and energy source (7-8, 29). To determine whether the strain can also grow by

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using the two other types of organic compounds widely found in nature, amino acids and

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organic acids, the growth of G. oxydans 621H on 19 proteinaceous amino acids and 24

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organic acids was investigated. D-Glucose and 1,2-propanediol were used as positive

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controls. As shown in Fig. 1A, after cultured for 24 h, the OD600 nm increased by more

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than 0.5 when G. oxydans 621H was grown on D-glucose and 1,2-propanediol, whereas

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no obvious growth was observed on proteinaceous amino acids, when compared with the

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growth of the negative control. With regard to the 24 organic acids used in this study

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(including glyoxylate; common hydroxy acids such as glycolate, D-/L-lactate, and

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D-/L-mandelate;

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oxalate, and citrate), only lactate, particularly D-lactate, supported the growth of G.

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oxydans 621H obviously (Fig. 1B).

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serves as an energy source to support growth of G. oxydans 621H. G.

and other common mono-, di-, and tri-carboxylic acids such as formate,

The robust growth of G. oxydans 621H on D-lactate (Fig. 2A) was accompanied by

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the rapid consumption of D-lactate (Fig. 2B) and the accumulation of pyruvate and

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acetate (Fig. 2C and 2D), indicating that D-lactate was first oxidized to pyruvate during

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the growth of G. oxydans 621H on D-lactate. However, when G. oxydans 621H was

249

cultured on pyruvate, little growth was detected when compared with the control. This

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suggests that the energy obtained from the oxidation of D-lactate to pyruvate is essential

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for the growth of G. oxydans 621H on D-lactate.

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G. oxydans 621H contains two D-lactate oxidizing enzymes. One mole of D-lactate provides two moles of electrons for the respiratory chain during the redox process (9). 11

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The electrochemical proton gradient generated by the electron transport chain in G.

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oxydans 621H is used to generate ATP via an F1F0-type ATP synthase (1). D-Lactate, but

256

not pyruvate, supported the growth of G. oxydans 621H, implying that the energy

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obtained from the oxidation of D-lactate to pyruvate was crucial for the growth of G.

258

oxydans 621H on D-lactate. To understand the D-lactate oxidation process, the D-lactate

259

oxidizing activity of G. oxydans 621H was assayed. G. oxydans 621H cells cultured in

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Y-S medium were broken using a high press cracker to obtain a crude cell extract, and the

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activities of iLDHs in G. oxydans 621H were assessed using MTT as an electron acceptor

262

and D-lactate or L-lactate as the substrate. As shown in Table 3, both D-iLDH and L-iLDH

263

activities were detected. The activity towards D-lactate was approximately 8-fold higher

264

than the activity towards L-lactate. After ultracentrifugation, the major D- and L-iLDH

265

activities were found in the membrane fractions, but a low D-iLDH activity remained in

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the supernatant (Table 3). Thus, in addition to a membrane-bound D-iLDH, at least one

267

soluble D-iLDH appeared to be present in the cytoplasm.

268

GOX1253 and GOX2071 are membrane-bound and soluble D-lactate oxidizing

269

enzymes, respectively. Two putative D-iLDHs (GOX1253 and GOX2071) have been

270

annotated in the genome of G. oxydans 621H (1). Comparative genomics analyses were

271

performed to compare their protein sequences with those of well-characterized D-iLDHs

272

(9, 30-32). GOX1253 shows high identity to D-iLDHs from E. coli (YP_490372.1, 55.2%

273

identity) and C. glutamicum (NP_600129.1, 46.4% identity) and low identity to D-iLDHs

274

from Archaeoglobus fulgidus (NP_069230.1, 14.3% identity) and Shewanella oneidensis

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(NP_717138.2, 8.2% identity). Comparatively, GOX2071 shows relatively high identity

276

to D-iLDH from A. fulgidus (28.2% identity) and low identity to D-iLDHs from E. coli

277

(12.4% identity), C. glutamicum (13.6% identity), and S. oneidensis (8.2% identity). 12

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To determine the intracellular location of the two putative D-iLDHs in G. oxydans

279

621H, the single and double mutants of GOX1253 and GOX2071, and their respective

280

complemented strains were constructed. The crude extracts were ultracentrifuged, and the

281

D-lactate

282

assayed. As shown in Table 4, no D-iLDH activity was found in the ultracentrifugation

283

supernatant or in the membrane fractions of G. oxydans ΔGOX1253 ΔGOX2071, which

284

indicated that there is no other D-iLDH in G. oxydans 621H. For G. oxydans ΔGOX2071,

285

activity was detected in the membrane fractions, while activity was only detected in the

286

ultracentrifugation supernatant of G. oxydans ΔGOX1253. These results suggest that

287

GOX1253 is a membrane-bound protein, while GOX2071 localizes in the cytoplasm,

288

which is rare for D-iLDHs. These findings were confirmed by the specific activities of G.

289

oxydans ΔGOX1253 ΔGOX2071 complemented with GOX1253 or GOX2071. The

290

unexpected activity in the ultracentrifugation supernatant of the GOX1253 complemented

291

strain was likely attributable to the fact that the overexpressed GOX1253 formed micelles

292

during lysis that did not sediment with the membrane fraction upon ultracentrifugation

293

(33).

oxidizing activities in the resultant supernatants and membrane fractions were

294

Purification and characterization of GOX1253. To characterize GOX1253, the

295

gene encoding GOX1253 was cloned and overexpressed in E. coli C43 (DE3), and the

296

protein with His-tag at its C-terminus was purified using a HisTrap column (Fig. 3A). In

297

size-exclusion chromatography analysis, the size of active GOX1253 calculated

298

according to its retention volume (11.660 ml) was 280.3 kDa (Fig. 3B) and the weight of

299

its monomer was calculated as 67.6 kDa according to its deduced amino acid sequence.

300

Thus, purified recombinant GOX1253 showed a polymerization degree of 4.15,

301

indicating that active GOX1253 exists as a tetramer. The purified GOX1253 solution was 13

302

light yellow with typical flavin absorption peaks around 460 nm and 380 nm, indicating

303

the presence of flavin. The flavin released from denatured GOX1253 by boiling migrated

304

identically with the FAD standard in HPLC, confirming that the flavin was FAD (Fig. 3C).

305

The estimated ratio of GOX1253 monomer to FAD was 2.24 ± 0.02 (Fig. S1). Therefore,

306

the active GOX1253 enzyme appears to contain one FAD per two subunits.

307

To analyze the product of the GOX1253 catalyzed D-lactate dehydrogenation, the

308

reaction solution containing 50 mM Tris-HCl (pH 7.4), 20 mM D-lactate, 2 U/ml purified

309

GOX1253, and 1 mM MTT was incubated aerobically at 30°C and 180 rpm for 30 min,

310

boiled to terminate the reactions and then subjected to HPLC analysis. The reactions with

311

denatured GOX1253 or without MTT addition were also conducted under identical

312

conditions (Fig. 3D). The results showed that pyruvate was obtained from the reaction

313

solution containing 1 mM MTT as an electron acceptor, whereas little pyruvate was found

314

in the reaction solution without MTT. Thus, GOX1253 is a typical D-iLDH that needs

315

artificial electron acceptors for D-lactate oxidation in vitro.

316

Purification and characterization of GOX2071. The gene encoding GOX2071

317

was also cloned and overexpressed in E. coli C43 (DE3). The fold purification and yields

318

of GOX2071 are shown in Table S1. Purified GOX2071 was obtained successively using

319

a Source 30Q column and a HisTrap column (Fig. 4A). In size-exclusion chromatography

320

analysis, the size of active GOX2071 calculated according to its retention volume (13.466

321

ml) was 119.0 kDa (Fig. 4B) while the weight of its monomer was calculated as 54.0 kDa.

322

Thus, active GOX2071 showed a polymerization degree of 2.20, indicating that it exists

323

as a dimer. Purified GOX2071 had an intense yellow color with typical flavin absorption

324

peaks around 460 nm and 380 nm, indicating that GOX2071 is also a flavoprotein. The

325

flavin released from denatured GOX2071 migrated identically with the FAD standard in 14

326

HPLC, confirming that the flavin was FAD (Fig. 4C). The estimated ratio of GOX2071

327

monomer to FAD was 1.15 ± 0.00 (Fig. S1). Thus, active GOX2071 contains one FAD

328

per subunit.

329

The catalytic properties of GOX2071 were investigated in the reaction solutions

330

containing 2 U/ml GOX2071, as described above for GOX1253. In the absence of MTT,

331

when O2 was used as the sole electron acceptor, 1.99 mM pyruvate and 9.52 mM acetate

332

were detected. In comparison, 3.90 mM pyruvate and 6.58 mM acetate were obtained

333

when MTT was present (Fig. 4D). The results indicated that GOX2071 used O2 as a

334

direct electron acceptor, which is consistent with the properties of an oxidase. The higher

335

amount of pyruvate (3.90 mM) and lower amount of acetate (6.58 mM) obtained when

336

MTT was added might reflect competition of MTT with O2 as the direct electron

337

acceptor.

338

Difference between GOX1253 and GOX2071. The substrate specificities of

339

GOX1253 and GOX2071 were determined with 10 mM 2-hydroxy acids with different

340

side chains using MTT as the electron acceptor. Both GOX1253 and GOX2071 preferred

341

D-2-hydroxy

342

22.31 U/mg, GOX1253 had narrow substrate specificity and incomplete stereoselectivity

343

for the D-isomer (i.e., low but detectable activity for L-lactate) (Fig. 5A). Comparatively,

344

GOX2071 showed a relatively low specific activity for D-lactate (0.35 U/mg), but had

345

broad substrate specificity. Furthermore, GOX2071 had higher specific activity towards

346

long-chain D-2-hydroxy acids (such as D-2-hydroxybutyrate, DL-2-hydroxyvalerate, and

347

DL-2-hydroxyisocaproate),

348 349

acids. Although the specific activity of GOX1253 for D-lactate reached

up to three times its activity towards D-lactate (Fig. 5A).

With regard to different electron acceptors, GOX1253 and GOX2071 showed distinct preferences (Table 5). GOX1253 preferred to use dichlorophenol-indophenol 15

350

(DCPIP) as an electron acceptor and showed moderate activity using MTT and

351

K3[Fe(CN)6]. However, it had extremely low specific activity when using O2 as an

352

electron acceptor (about 477 times lower than that with DCPIP). Comparatively,

353

GOX2071 showed nearly equal specific activity with DCPIP, MTT, and O2 as electron

354

acceptors.

355

To confirm the classification of GOX1253 and GOX2071, their abilities to use O2 as

356

a direct electron acceptor were assessed using a Clark-type oxygen electrode as described

357

in a previous study (34). Purified enzyme (0.016 U/ml) was added to a reaction chamber

358

containing 50 mM Tris-HCl (pH 7.4) and 20 mM D-lactate. Little O2 consumption was

359

detected after the addition of GOX1253 for 2 min, while rapid O2 consumption was

360

observed when PMS was added (Fig. 5B), indicating that GOX1253 is a typical D-iLDH.

361

O2 consumption was noticeable immediately following the addition of GOX2071, and the

362

rate of O2 consumption did not vary after the addition of PMS (Fig. 5B), suggesting that

363

GOX2071 can use O2 as a direct electron acceptor to oxidize D-lactate. GOX2071

364

appears to be a novel type of D-iLDH, a D-lactate oxidase.

365

Catalytic mechanism of GOX2071. L-Lactate oxidase has been studied and used in

366

biosensors for many years (16, 35). The enzyme catalyzes the oxidation of L-lactate into

367

pyruvate and hydrogen peroxide using O2 as a direct electron acceptor. In order to

368

elucidate the catalytic mechanism of GOX2071, five aerobic reaction systems were

369

assessed at 30°C for 30 min, as shown in Table 6. Catalase was added to the system to

370

transform hydrogen peroxide, a possible product, into H2O and O2. Pyruvate and acetate

371

were found as products in system 1, which contained GOX2071 and D-lactate. When

372

catalase was added, as in system 2, pyruvate rather than acetate became the main product,

373

which suggested that catalase stopped the oxidation of pyruvate to acetate by catalyzing 16

374

the decomposition of H2O2. System 3 showed that GOX2071 could not convert pyruvate

375

to acetate, which confirmed the previous supposition. Systems 4 and 5 were used to

376

demonstrate that H2O2 rather than GOX2071 oxidized pyruvate to acetate. The results

377

showed that artificially added H2O2 oxidized pyruvate to acetate without the need for an

378

enzyme and that catalase stopped the oxidization. Thus, the results indicated that

379

GOX2071, a D-lactate oxidase, catalyzed the oxidation of D-lactate to pyruvate and H2O2

380

and that the generated H2O2 oxidized pyruvate to acetate, as schemed in the following

381

reactions.

382 383

The kinetic parameters of purified GOX2071 were evaluated using a Clark-type

384

oxygen electrode. The estimated apparent Km and Vmax of GOX2071 towards D-lactate

385

were 3.62 ± 0.53 mM and 0.52 ± 0.05 U/mg, respectively (Table 7). The data is similar to

386

that reported for L-lactate oxidase from Geotrichum candidum (Km 3.6 mM, Vmax 1.26

387

U/mg) (36).The estimated apparent Km and Vmax for O2, the other substrate, were 0.16 ±

388

0.01 mM and 0.97 ± 0.03 U/mg, respectively (Table 7).

389

Physiological functions of GOX1253 and GOX2071. To clarify the roles of

390

GOX1253 and GOX2071 in D-lactate utilization in vivo, the D-lactate oxidizing capacity

391

of whole cells of G. oxydans 621H wild-type and mutant strains were tested. The reaction

392

broths contained 20 mM phosphate buffer (pH 7.0), 85 mM D-lactate, and 100 g/l wet

393

cells at 30°C and 180 rpm. Rapid consumption of D-lactate by G. oxydans 621H wild-type

394

cells was observed, whereas almost no perceivable D-lactate consumption was found in

395

reaction broths containing G. oxydans ΔGOX1253 or G. oxydans ΔGOX1253 ΔGOX 2071

396

cells (Fig. S2). These results indicated that the cells lost their ability to oxidize D-lactate 17

397

when GOX1253 was deleted. In contrast, the deletion of GOX2071 had no apparent

398

effect on the D-lactate oxidation in G. oxydans 621H (Fig. S2).

399

To investigate the physiological roles of GOX1253 and GOX2071 in the growth of G.

400

oxydans 621H on D-lactate, G. oxydans 621H and its derivatives were cultivated in YE-P

401

medium supplemented with 50 mM D-lactate. As expected, all G. oxydans 621H-derived

402

strains containing GOX1253 were capable of utilizing D-lactate to support their growth,

403

while strains lacking GOX1253 were not (Fig. 6A and Fig. 6B). These results strongly

404

suggest that GOX1253 is the key enzyme that supports the growth of G. oxydans 621H on

405

D-lactate.

406

oxidation by GOX2071, GOX2071 did not contribute to the D-lactate-driven growth of G.

407

oxydans 621H.

Because no usable energy or carbon source was produced from D-lactate

408 409

DISCUSSION

410

In this study, D-lactate was identified as one of a few available organic acids that can act

411

as an energy source to support the growth of G. oxydans 621H. The oxidized product,

412

pyruvate, did not support the growth of G. oxydans 621H. The energy obtained from

413

D-lactate

414

on D-lactate. Consequently, the enzymes responsible for D-lactate oxidation in G. oxydans

415

621H and their mechanisms were the focus of this study. Enzyme activity analysis of G.

416

oxydans 621H wild-type and mutants identified two D-lactate oxidizing enzymes,

417

GOX1253 and GOX2071, with differential subcellular distributions (Table 4). GOX1253

418

is located on the cytoplasmic membrane of G. oxydans 621H. In contrast, GOX2071 is a

419

soluble protein located in the cytoplasm.

420

oxidation would be the major driving force for the growth of G. oxydans 621H

Comparison of the GOX1253 sequence with the GenBank database using the 18

421

BLAST program showed that the predicted FAD-binding domain of GOX1253 is located

422

near the N-terminus of the protein, while the C-terminus contains a predicted

423

membrane-bound lactate dehydrogenase domain. The predicted domains of GOX1253 are

424

similar to that of respiratory D-iLDH from E. coli (9). The BLASTP result of GOX2071

425

also revealed an FAD-binding domain at the N-terminus, but an FAD-oxidase domain

426

was found at the C-terminus. Furthermore, phylogenetic analyses of GOX1253 and

427

GOX2071 homologs (Fig. 7 and Table S2) show that the enzymes belong to two distinct

428

clusters. GOX1253 is strongly related to E. coli D-iLDH and the D-iLDHs from

429

Proteobacteria that use quinone as a natural electron acceptor; they form a distinct cluster

430

relative to the other D-iLDHs. D-iLDHs similar to GOX2071, which contain only the

431

FAD-binding and FAD-oxidase domains, are found in all three domains of life, unlike

432

quinone-dependent D-iLDHs, which are only found in Bacteria. Moreover, very strong

433

bootstrap support indicates that GOX2071 has a closer phylogenetic relationship to

434

D-iLDHs

from Animalia than to those from Fungi and Plantae (37).

435

Among the reported bacteria, only three strains (Photobacterium profundum,

436

Propionibacterium acnes, and Brevibacterium linens) possess two putative genes

437

encoding D-iLDHs, dld (similar to E. coli D-iLDH) and dld-II (similar to SO_1521 of S.

438

oneidensis MR-1) (32), but their respective functions in these strains have not been

439

reported. In this study, two enzymes for D-lactate oxidation were identified in G. oxydans

440

621H, and their catalytic characteristics, kinetic properties, functions, and physiological

441

significance were investigated. To date, G. oxydans 621H is the only bacterium in which

442

two enzymes have been shown experimentally to oxidize D-lactate. GOX1253 contributes

443

to the rapid oxidization of D-lactate to pyruvate on the membrane (Fig. 6). Subsequently,

444

the synthesized pyruvate is likely to be converted to acetate (Fig. 2D) by pyruvate 19

445

decarboxylase (GOX1081) and aldehyde dehydrogenase (GOX2018) acting sequentially

446

in the cytoplasm (29, 38). Indeed, no alternative pathway for the production of acetate

447

from pyruvate appears to exist in G. oxydans 621H, which lacks phosphotransacetylase

448

(EC 2.3.1.8), acetate kinase (EC 2.7.2.1), and pyruvate:quinone oxidoreductase (EC

449

1.2.2.2) (29). The physiological function of GOX2071, which displayed no perceptible

450

activity in D-lactate oxidation in vivo (Fig. 6), was not clarified in this study. Nevertheless,

451

GOX2071 might also play a role in the oxidation of D-lactate to acetate in the cytoplasm.

452

On the basis of the results and suppositions described above, we propose a model for

453

D-lactate

454

D-lactate,

455

reported. However, L-lactate permease (LldP) and glycolate permease (GlcA) of E. coli

456

can transport D-lactate (39). When searching the G. oxydans 621H genome, GOX2098

457

was found to exhibit high protein sequence identities to E. coli LldP (31.4%) and GlcA

458

(50.5%), suggesting that GOX2098 might transport D-lactate in G. oxydans 621H.

459

oxidation in G. oxydans 621H, as shown in Fig. 8. Regarding the transport of no proteins specific for the transport of D-lactate into the cytoplasm have been

D-Lactate

was used as an energy source in G. oxydans 621H (Fig. 2), unlike in E.

460

coli and C. glutamicum (9, 11). The product of D-lactate oxidation by E. coli or C.

461

glutamicum D-iLDH, pyruvate, is further oxidized to acetyl-CoA by the pyruvate

462

dehydrogenase complex. Then, acetyl-CoA enters into the TCA cycle to provide carbon

463

and energy resources for bacterial growth. However, the TCA cycle and EMP pathway

464

are incomplete in G. oxydans because of the lack of succinate dehydrogenase and

465

6-phosphofructokinase (1). Furthermore, G. oxydans 621H cannot produce C6-sugars via

466

gluconeogenesis; no open reading frames that potentially encode a phosphoenolpyruvate

467

synthase or other phosphoenolpyruvate-synthesizing enzymes have been identified in its

468

genome (1). Therefore, pyruvate derived from D-lactate cannot enter into the TCA cycle 20

469

for further metabolism or for production of C6-sugars via gluconeogenesis, and the

470

accumulated pyruvate must enter a non-energy generation pathway with acetate as the

471

final product. Therefore, the D-lactate-driven growth of G. oxydans depends on the limited

472

energy derived from the oxidation of D-lactate to pyruvate via membrane-bound

473

GOX1253.

474

The crystal structure of respiratory D-iLDH from E. coli has been solved, and its

475

physiological features and properties have been extensively studied (9-10). GOX1253 is

476

also a membrane-bound protein, whose over-expression and purification may be

477

relatively difficult. Thus, E. coli C43 (DE3), a mutant of E. coli BL21 (DE3) suitable for

478

membrane-bound protein expression (40), was used for the over-expression of GOX1253.

479

Purified GOX1253 exhibits similar catalytic characteristics to E. coli D-iLDH, such as

480

high activity towards D-lactate, narrow substrate specificity, incomplete stereoselectivity

481

for the D-isomer (i.e., low but detectable activity towards L-lactate), and a requirement for

482

an artificial electron acceptor for the continuous oxidation of D-lactate (10). Given the

483

rather simple respiratory chain in G. oxydans 621H (1), together with the fact that the

484

energy for growth comes only from D-lactate oxidation via GOX1253, we can speculate

485

on the relationship between GOX1253 and the respiratory chain in cellular energy

486

provision. Membrane-bound GOX1253 transfers two moles of electrons to ubiquinone

487

when oxidizing one mole of D-lactate. Two quinol oxidases, cytochrome bo3 and bd, then

488

catalyze the transfer of electrons from the reduced ubiquinone to molecular oxygen,

489

which is accompanied by the generation of proton motive force (1-2, 41). Subsequently,

490

the electrochemical proton gradient is used to generate ATP via an F1F0-type ATP

491

synthase (2).

492

Unlike other reported D-iLDHs, GOX2071 aerobically oxidizes D-lactate in the 21

493

absence of an artificial electron acceptor (Fig. 5B). This indicates that GOX2071 is the

494

first experimentally confirmed D-lactate oxidase (13). In addition, GOX2071 exhibits

495

high stereoselectivity for D-isomers and broad substrate specificity for D-2-hydroxy acids

496

with different carbon chain lengths (Fig. 5A). Considering its ability to utilize O2 as the

497

direct electron acceptor, GOX2071 has potential for use in the resolution of racemic

498

2-hydroxy acids to produce optically pure L-2-hydroxy acids, which are widely used as

499

pharmaceutical intermediates and platform chemical compounds.

500

D-Lactate

is metabolized in the human body to a limited extent, and its abnormal

501

accumulation causes D-lactic acidosis, a syndrome accompanied by recurrent episodes of

502

encephalopathy and metabolic acidosis (42-44). The clinical diagnosis of D-lactic acidosis

503

must be confirmed by measuring the D-lactate level at the time of an acute attack (42).

504

However, routine lactate determinations only measure L-lactate. Thus, a rapid detection

505

method, such as the D-lactate biosensor, specific for D-lactate is urgently needed (42-43).

506

The widely used L-lactate biosensor is usually based on the amperometric detection of

507

H2O2 released from L-lactate oxidation via L-lactate oxidase (16, 45). Unlike

508

membrane-bound D-iLDHs, which require detergent for stability (13), soluble GOX2071

509

is favorable for immobilization to the electrode surface. On the other hand, the H2O2

510

production capacity of GOX2071 makes it suitable for biosensors, similar to L-lactate

511

oxidase.

512

In summary, our study clarifies the unusual mechanism for the D-lactate-dependent

513

growth of G. oxydans, in which D-lactate is used as an energy source. GOX1253 is the

514

key enzyme providing energy for the growth of G. oxydans 621H, via one-step oxidation

515

of D-lactate accompanied by electron transfer to a quinone pool. In contrast, GOX2071

516

oxidizes D-lactate using O2 as a direct electron acceptor without usable energy production; 22

517

thus, it does not contribute to the D-lactate-driven growth of the strain. Furthermore, the

518

first identified D-lactate oxidase, GOX2071, together with its homologs, will increase

519

interest in the development of new biocatalysts for racemic 2-hydroxy acid resolution and

520

new D-lactate biosensors.

521 522

ACKNOWLEDGMENTS

523

The work was supported by the National Natural Science Foundation of China (31270856

524

and 31170052) and the Chinese National Program for High Technology Research and

525

Development (2014AA021206). We thank Prof. Uwe Deppenmeier (Universität Bonn)

526

for providing the expression vector, pBBR1p264. The authors declare no conflict of

527

interest.

528 529

REFERENCES

530

1.

Prust C, Hoffmeister M, Liesegang H, Wiezer A, Fricke WF, Ehrenreich A,

531

Gottschalk G, Deppenmeier U. 2005. Complete genome sequence of the acetic

532

acid bacterium Gluconobacter oxydans. Nat Biotechnol 23:195–200.

533

http://dx.doi.org/10.1038/nbt1062.

534

2.

Deppenmeier U, Ehrenreich A. 2009. Physiology of acetic acid bacteria in light

535

of the genome sequence of Gluconobacter oxydans. J Mol Microbiol Biotechnol

536

16:69–80. http://dx.doi.org/10.1159/000142895.

537

3.

De Muynck C, Pereira CSS, Naessens M, Parmentier S, Soetaert W,

538

Vandamme EJ. 2007. The genus Gluconobacter oxydans: Comprehensive

539

overview of biochemistry and biotechnological applications. Crit Rev Biotechnol

540

27:147–171. http://dx.doi.org/10.1080/07388550701503584. 23

541

4.

542 543

Gupta A, Singh VK, Qazi GN, Kumar A. 2001. Gluconobacter oxydans:its biotechnological applications. J Mol Microbiol Biotechnol 3:445–456.

5.

Deppenmeier U, Hoffmeister M, Prust C. 2002. Biochemistry and

544

biotechnological applications of Gluconobacter strains. Appl Microbiol

545

Biotechnol 60:233–242. http://dx.doi.org/10.1007/s00253-002-1114-5.

546

6.

Rollini M, Manzoni M. 2005. Bioconversion of D-galactitol to tagatose and

547

dehydrogenase activity induction in Gluconobacter oxydans. Process Biochem

548

40:437–444. http://dx.doi.org/10.1016/j.procbio.2004.01.028.

549

7.

Gao C, Zhang W, Huang Y, Ma C, Xu P. 2012. Efficient conversion of

550

1,2-butanediol to (R)-2-hydroxybutyric acid using whole cells of Gluconobacter

551

oxydans. Bioresour Technol 115:75–78.

552

http://dx.doi.org/10.1016/j.biortech.2011.11.009.

553

8.

Peters B, Mientus M, Kostner D, Junker A, Liebl W, Ehrenreich A. 2013.

554

Characterization of membrane-bound dehydrogenases from Gluconobacter

555

oxydans 621H via whole-cell activity assays using multideletion strains. Appl

556

Microbiol Biotechnol 97:6397–6412.

557

http://dx.doi.org/10.1007/s00253-013-4824-y.

558

9.

Dym O, Pratt EA, Ho C, Eisenberg D. 2000. The crystal structure of D-lactate

559

dehydrogenase, a peripheral membrane respiratory enzyme. Proc Natl Acad Sci U

560

S A 97:9413–9418. http://dx.doi.org/10.1073/pnas.97.17.9413.

561

10.

Kohn LD, Kaback HR. 1973. Mechanisms of active transport in isolated

562

bacterial membrane vesicles: XV. Purification and properties of the

563

membrane-bound D-lactate dehydrogenase from Escherichia coli. J Biol Chem

564

248:7012–7017. 24

565

11.

Kato O, Youn JW, Stansen KC, Matsui D, Oikawa T, Wendisch VF. 2010.

566

Quinone-dependent D-lactate dehydrogenase Dld (Cg1027) is essential for growth

567

of Corynebacterium glutamicum on D-lactate. BMC Microbiol 10:321.

568

http://dx.doi.org/10.1186/1471-2180-10-321.

569

12.

Ma C, Gao C, Qiu J, Hao J, Liu W, Wang A, Zhang Y, Wang M, Xu P. 2007.

570

Membrane-bound L- and D-lactate dehydrogenase activities of a newly isolated

571

Pseudomonas stutzeri strain. Appl Microbiol Biotechnol 77:91–98.

572

http://dx.doi.org/10.1007/s00253-007-1132-4.

573

13.

Jiang T, Gao C, Ma C, Xu P. 2014. Microbial lactate utilization: enzymes,

574

pathogenesis, and regulation. Trends Microbiol 22:589–599.

575

http://dx.doi.org/10.1016/j.tim.2014.05.008.

576

14.

Zheng Z, Sheng B, Ma C, Zhang H, Gao C, Su F, Xu P. 2012. Relative catalytic

577

efficiency of ldhL- and ldhD-encoded products is crucial for optical purity of

578

lactic acid produced by Lactobacillus strains. Appl Environ Microbiol

579

78:3480–3483. http://dx.doi.org/10.1128/AEM.00058-12.

580

15.

Wang L, Cai Y, Zhu L, Guo H, Yu B. 2014. Major role of NAD-dependent

581

lactate dehydrogenases in high optically pure L-lactic acid production by

582

thermophilic Bacillus coagulans. Appl Environ Microbiol.

583

http://dx.doi.org/10.1128/AEM.01864-14.

584

16.

Rassaei L, Olthuis W, Tsujimura S, Sudhölter EJR, van den Berg A. 2014.

585

Lactate biosensors: current status and outlook. Anal Bioanal Chem 406:123–137.

586

http://dx.doi.org/10.1007/s00216-013-7307-1.

587 588

17.

Burdick BA, Schaeffer JR. 1987. Co-immobilized coupled enzyme systems on nylon mesh capable of gluconic and pyruvic acid production. Biotechnol Lett 25

589 590

9:253–258. http://dx.doi.org/10.1007/BF01027159. 18.

591 592

Xu P, Qiu J, Gao C, Ma C. 2008. Biotechnological routes to pyruvate production. J Biosci Bioeng 105:169–175. http://dx.doi.org/10.1263/jbb.105.169.

19.

Su W, Chang Z, Gao K, Wei D. 2004. Enantioselective oxidation of racemic

593

1,2-propanediol to D-(−)-lactic acid by Gluconobacter oxydans. Tetrahedron:

594

Asymmetry 15:1275–1277. http://dx.doi.org/10.1016/j.tetasy.2004.03.009.

595

20.

Schäfer A, Tauch A, Jäger W, Kalinowski J, Thierbach G, Pühler A. 1994.

596

Small mobilizable multi-purpose cloning vectors derived from the Escherichia

597

coli plasmids pK18 and pK19: selection of defined deletions in the chromosome

598

of Corynebacterium glutamicum. Gene 145:69–73.

599

http://dx.doi.org/10.1016/0378-1119(94)90324-7.

600

21.

Peters B, Junker A, Brauer K, Mühlthaler B, Kostner D, Mientus M, Liebl W,

601

Ehrenreich A. 2013. Deletion of pyruvate decarboxylase by a new method for

602

efficient markerless gene deletions in Gluconobacter oxydans. Appl Microbiol

603

Biotechnol 97:2521–2530. http://dx.doi.org/10.1007/s00253-012-4354-z.

604

22.

Kawai S, Goda-Tsutsumi M, Yakushi T, Kano K, Matsushita K. 2013.

605

Heterologous overexpression and characterization of a flavoprotein-cytochrome c

606

complex fructose dehydrogenase of Gluconobacter japonicus NBRC3260. Appl

607

Environ Microbiol 79:1654–1660. http://dx.doi.org/10.1128/aem.03152-12.

608

23.

Kallnik V, Meyer M, Deppenmeier U, Schweiger P. 2010. Construction of

609

expression vectors for protein production in Gluconobacter oxydans. J Biotechnol

610

150:460–465. http://dx.doi.org/10.1016/j.jbiotec.2010.10.069.

611 612

24.

Dumon-Seignovert L, Cariot G, Vuillard L. 2004. The toxicity of recombinant proteins in Escherichia coli: a comparison of overexpression in BL21(DE3), 26

613

C41(DE3), and C43(DE3). Protein Expr Purif 37:203–206.

614

http://dx.doi.org/10.1016/j.pep.2004.04.025.

615

25.

Markwell MAK, Haas SM, Bieber LL, Tolbert NE. 1978. A modification of the

616

Lowry procedure to simplify protein determination in membrane and lipoprotein

617

samples. Anal Biochem 87:206–210.

618

http://dx.doi.org/10.1016/0003-2697(78)90586-9.

619

26.

Gao C, Jiang T, Dou P, Ma C, Li L, Kong J, Xu P. 2012. NAD-independent

620

L-lactate

621

stutzeri SDM. PLoS One 7:e36519.

622

http://dx.doi.org/10.1371/journal.pone.0036519.

623

27.

dehydrogenase is required for L-lactate utilization in Pseudomonas

Eppink MHM, Boeren SA, Vervoort J, van Berkel WJH. 1997. Purification

624

and properties of 4-hydroxybenzoate 1-hydroxylase (decarboxylating), a novel

625

flavin adenine dinucleotide-dependent monooxygenase from Candida

626

parapsilosis CBS604. J Bacteriol 179:6680–6687.

627

28.

Li L, Li K, Wang Y, Chen C, Xu Y, Zhang L, Han B, Gao C, Tao F, Ma C, Xu

628

P. 2015. Metabolic engineering of Enterobacter cloacae for high-yield production

629

of enantiopure (2R,3R)-2,3-butanediol from lignocellulose-derived sugars. Metab

630

Eng 28:19-27. http://dx.doi.org/10.1016/j.ymben.2014.11.010.

631

29.

Krajewski V, Simić P, Mouncey NJ, Bringer S, Sahm H, Bott M. 2010.

632

Metabolic engineering of Gluconobacter oxydans for improved growth rate and

633

growth yield on glucose by elimination of gluconate formation. Appl Environ

634

Microbiol 76:4369–4376. http://dx.doi.org/10.1128/AEM.03022-09.

635 636

30.

Reed DW, Hartzell PL. 1999. The Archaeoglobus fulgidus D-lactate dehydrogenase is a Zn2+ flavoprotein. J Bacteriol 181:7580–7587. 27

637

31.

Stansen C, Uy D, Delaunay S, Eggeling L, Goergen JL, Wendisch VF. 2005.

638

Characterization of a Corynebacterium glutamicum lactate utilization operon

639

Induced during temperature-triggered glutamate production. Appl Environ

640

Microbiol 71:5920–5928. http://dx.doi.org/10.1128/aem.71.10.5920-5928.2005.

641

32.

Pinchuk GE, Rodionov DA, Yang C, Li X, Osterman AL, Dervyn E,

642

Geydebrekht OV, Reed SB, Romine MF, Collart FR, Scott JH, Fredrickson

643

JK, Beliaev AS. 2009. Genomic reconstruction of Shewanella oneidensis MR-1

644

metabolism reveals a previously uncharacterized machinery for lactate utilization.

645

Proc Natl Acad Sci U S A 106:2874–2879.

646

http://dx.doi.org/10.1073/pnas.0806798106.

647

33.

Reddy Pagala V, Park J, Reed DW, Hartzell PL. 2002. Cellular localization of

648

D-lactate

649

Archaea 1:95–104. http://dx.doi.org/10.1155/2002/297264.

650

34.

dehydrogenase and NADH oxidase from Archaeoglobus fulgidus.

Yuan H, Fu G, Brooks PT, Weber I, Gadda G. 2010. Steady-state kinetic

651

mechanism and reductive half-reaction of D-arginine dehydrogenase from

652

Pseudomonas aeruginosa. Biochemistry 49:9542–9550.

653

http://dx.doi.org/10.1021/bi101420w.

654

35.

655 656

Lockridge O, Massey V, Sullivan PA. 1972. Mechanism of action of the flavoenzyme lactate oxidase. J Biol Chem 247:8097–8106.

36.

Sztajer H, Wang W, Lünsdorf H, Stocker A, Schmid RD. 1996. Purification

657

and some properties of a novel microbial lactate oxidase. Appl Microbiol

658

Biotechnol 45:600–606. http://dx.doi.org/10.1007/s002530050736.

659 660

37.

Engqvist M, Drincovich MF, Flügge U-I, Maurino VG. 2009. Two D-2-hydroxy-acid

dehydrogenases in Arabidopsis thaliana with catalytic 28

661

capacities to participate in the last reactions of the methylglyoxal and β-oxidation

662

pathways. J Biol Chem 284:25026–25037.

663

http://dx.doi.org/10.1074/jbc.M109.021253.

664

38.

Richhardt J, Bringer S, Bott M. 2012. Mutational analysis of the pentose

665

phosphate pathway and the Entner-Doudoroff pathway in Gluconobacter oxydans

666

reveals improved growth of an Δedd Δeda deletion mutant on mannitol. Appl

667

Environ Microbiol 78:6975–6986. http://dx.doi.org/10.1128/AEM.01166-12.

668

39.

Núñez MF, Kwon O, Wilson TH, Aguilar J, Baldoma L, Lin ECC. 2002.

669

Transport of L-lactate, D-lactate, and glycolate by the LldP and GlcA membrane

670

carriers of Escherichia coli. Biochem Biophys Res Commun 290:824–829.

671

http://dx.doi.org/10.1006/bbrc.2001.6255.

672

40.

Miroux B, Walker JE. 1996. Over-production of proteins in Escherichia coli:

673

mutant hosts that allow synthesis of some membrane proteins and globular

674

proteins at high levels. J Mol Biol 260:289–298.

675

http://dx.doi.org/10.1006/jmbi.1996.0399.

676

41.

Richhardt J, Luchterhand B, Bringer S, Büchs J, Bott M. 2013. Evidence for a

677

key role of cytochrome bo3 qxidase in respiratory energy metabolism of

678

Gluconobacter oxydans. J Bacteriol 195:4210–4220.

679

http://dx.doi.org/10.1128/jb.00470-13.

680

42.

Vella A, Farrugia G. 1998. D-Lactic acidosis: pathologic consequence of

681

saprophytism. Mayo Clin Proc 73:451–456.

682

http://dx.doi.org/10.1016/S0025-6196(11)63729-4.

683 684

43.

Inoue Y, Shinka T, Ohse M, Kohno M, Konuma K, Ikawa H, Kuhara T. 2007. Changes in urinary level and configuration ratio of D-lactic acid in patients with 29

685

short bowel syndrome. J Chromatogr B 855:109–114.

686

http://dx.doi.org/10.1016/j.jchromb.2007.02.052.

687

44.

Reddy VS, Patole SK, Rao S. 2013. Role of probiotics in short bowel syndrome

688

in infants and children—a systematic review. Nutrients 5:679–699.

689

http://dx.doi.org/10.3390/nu5030679.

690

45.

Iwuoha EI, Rock A, Smyth MR. 1999. Amperometric L-lactate biosensors: 1.

691

Lactic acid sensing electrode containing lactate oxidase in a composite

692

poly-L-lysine matrix. Electroanalysis 11:367–373.

693

http://dx.doi.org/10.1002/(sici)1521-4109(199905)11:53.0.co;

694

2-0.

695

30

696

FIGURE LEGENDS

697

FIG 1 Comparison of the growth of G. oxydans 621H on different organic compounds. G.

698

oxydans 621H was cultured in YE-P medium supplemented with 50 mM of D-glucose,

699

1,2-propanediol, and 19 proteinaceous amino acids (A) or 24 organic acids (B),

700

respectively. Growth (∆OD600 nm) was calculated by the final OD reached at 24 h minus

701

the initial OD at 600 nm. A slight growth of G. oxydans 621H in YE-P medium (control)

702

might be caused by small amounts of utilizable organic compounds residing in the

703

inoculated cells, or provided by yeast extracts and peptone. All error bars represent the

704

standard deviations of at least three independent experiments.

705 706

FIG 2 The impacts of D-lactate and pyruvate on the growth of G. oxydans 621H in YE-P

707

medium. G. oxydans 621H was cultured in YE-P medium (as control) and YE-P medium

708

supplemented with 50 mM D-lactate or 50 mM pyruvate, respectively. Cell density (A),

709

D-lactate

710

the standard deviations of three independent experiments.

(B), pyruvate (C), and acetate (D) in mediums were assayed. Error bars indicate

711 712

FIG 3 Purification and characterization of GOX1253. (A) SDS-PAGE of expression and

713

purification steps of GOX1253. Lane M, molecular weight markers; lane 1, crude extract

714

of E. coli C43 (DE3) harboring pET25b; lane 2, crude extract of E. coli C43 (DE3)

715

harboring pET25b-GOX1253; lane 3, purified GOX1253 using a HisTrap column. (B)

716

Size-exclusion chromatography analysis of active GOX1253 using thyroglobulin (669

717

kDa), ferritin (440 kDa), aldolase (158 kDa), conalbumin (75 kDa), ovalbumin (43 kDa),

718

and ribonuclease A (13.7 kDa) as standard proteins. (C) HPLC analysis of flavin bounded

719

to GOX1253 using FAD, FMN and riboflavin as reference standards. (D) Analysis of the 31

720

product of the GOX1253 catalyzed D-lactate dehydrogenation. The reaction system (red

721

line) containing 50 mM Tris-HCl (pH 7.4), 20 mM D-lactate, 2 U/ml purified GOX1253,

722

and 1 mM MTT was incubated aerobically at 30°C and 180 rpm for 30 min and then

723

subjected to HPLC analysis. The reactions with denatured GOX1253 (black line) or

724

without MTT (green line) addition were also conducted under identical conditions.

725 726

FIG 4 Purification and characterization of GOX2071. (A) SDS-PAGE of expression and

727

purification steps of GOX2071. Lane M, molecular weight markers; lane 1, crude extract

728

of E. coli C43 (DE3) harboring pET25b; lane 2, crude extract of E. coli C43 (DE3)

729

harboring pET25b-GOX2071; lane 3, partially purified GOX2071 using a Source 30Q

730

column; lane 4, purified GOX2071 using a HisTrap column. (B) Size-exclusion

731

chromatography analysis of GOX2071 as described in Fig. 3B. (C) HPLC analysis of

732

flavin bounded to GOX2071 using FAD, FMN and riboflavin as reference standards. (D)

733

Analysis of the product of the GOX2071 catalyzed D-lactate dehydrogenation. Three

734

reactions with purified GOX2071 of 2 U/ml were conducted as described in Fig. 3D and

735

then subjected to HPLC analysis.

736 737

FIG 5 Difference between GOX1253 and GOX2071 on substrate specificity and

738

reactivity with molecular oxygen. (A) Specific activities of GOX1253 and GOX2071

739

towards sodium salts of different 2-hydroxy acids (50 mM) with MTT as the electron

740

acceptor. All error bars indicate the standard deviations of three independent experiments.

741

(B) D-Lactate-dependent oxygen consumption of GOX1253 and GOX2071 assayed by a

742

Clark-type oxygen electrode. The assay mixture contained 50 mM Tris-HCl (pH 7.4) and

743

20 mM D-lactate at 30°C at 900 rpm. After monitoring the background for about 1 min, 32

744

GOX1253 or GOX2071 (0.016 U/ml) was added and the oxygen traces were monitored.

745

Later (about 2 min), 0.4 mM PMS was added, and the oxygen traces were monitored for

746

another 2 min. Addition of PMS solution (air-saturated) would cause a slight increase of

747

oxygen concentration in reaction mixture as indicated.

748 749

FIG 6 Identification of the roles of GOX1253 and GOX2071 in the D-lactate-dependent

750

growth of G. oxydans 621H. The G. oxydans 621H (WT) and its derivatives were cultured

751

in YE-P medium supplemented with 50 mM D-lactate and the changes of cell density (A)

752

and D-lactate concentration (B) were assayed. Error bars indicate the standard deviations

753

of three independent experiments.

754 755

FIG 7 Phylogenetic analyses of GOX2071 and GOX1253. Proteins including

756

quinone-dependent D-iLDHs similar to GOX1253, Fe-S clusters containing D-iLDHs and

757

the D-iLDHs similar to GOX2071 in the three domains of life are listed in Table S2. The

758

phylogenetic tree was constructed by using MEGA software program (version 5.10) via

759

neighbor-joining and bootstrap method for 1000 replications with Poisson correction. The

760

asterisk marked that the bacteria do not belong to the same phylum with their neighbours.

761

Mastigocoleus testarum belongs to Cyanobacteria; Flavobacterium saliperosum belongs

762

to Bacteroidetes; and Corynebacterium glutamicum belongs to Actinobacteria. The scale

763

at the bottom indicates levels of amino acid sequence divergence.

764 765

FIG 8 Proposed model for D-lactate oxidation in G. oxydans 621H. The intracellular

766

oxidation of D-lactate can be catalyzed by both GOX1253 and GOX2071. GOX2071

767

oxidizes D-lactate directly via O2 in a way that GOX2071 does not provide any usable 33

768

energy for cells. GOX1253 contributes a lot to oxidize D-lactate to pyruvate rapidly on

769

the membrane, which is accompanied by the transport of electrons to the ubiquinone

770

pools to provide energy for the strain. Subsequently, pyruvate is converted to acetate by

771

pyruvate decarboxylase (GOX1081) and aldehyde dehydrogenase (GOX2018) acting

772

sequentially in the cytoplasm. D-Lactate might be transported via the lactate permease

773

(LldP) encoded by GOX2098.

774

34

775

TABLE 1 Strains and plasmids used in this study Strain G. oxydans 621H G. oxydans ΔGOX1253 G. oxydans ΔGOX2071 G. oxydans ΔGOX1253 ΔGOX2071 G. oxydans GOX1253 complement G. oxydans GOX2071 complement E. coli DH5α E. coli HB101 E. coli C43 (DE3) Plasmid pRK2013 pET25b pET25b-GOX1253 pET25b-GOX2071 pK18mobsacB pK18mobsacB-ΔGOX1253 pK18mobsacB-ΔGOX2071

776

Characteristics G. oxydans DSM2343, wild type, Cefr GOX1253 deletion strain of G. oxydans 621H, Cefr GOX2071 deletion strain of G. oxydans 621H, Cefr GOX1253 and GOX2071 deletion strain of G. oxydans 621H, Cefr G. oxydans ΔGOX1253 ΔGOX2071 harboring pBBR1p264-GOX1253, Cefr, Kmr G. oxydans ΔGOX1253 ΔGOX2071 harboring pBBR1p264-GOX2071, Cefr, Kmr F– φ80lacZ∆M15 ∆(lacZYA-argF)U169 recA1 endA1 hsdR17(rK–, mK+) phoA supE44 thi-1 gyrA96 relA1 λ–, used for gene clone F– mcrB mrr hsdS20(rB–, mB–) recA13 supE44 ara14 galK2 lacY1 proA2 rpsL20(Smr) xy15 λ– leu mtl1, used for triparental mating as the helper strain Mutant of E. coli BL21 (DE3) for protein expression r

Vector mediates plasmid transfer; Km Vector for protein expression, Apr pET25b with GOX1253 gene of G. oxydans 621H, Apr pET25b with GOX2071 gene of G. oxydans 621H, Apr Allelic exchange vector, oriColE1 Mob+, lacZα, sacB，Kmr A pK18mobsacB derivative for deletion of GOX1253, Kmr A pK18mobsacB derivative for deletion of GOX2071, Kmr Expression vector with a strong promoter derived from pBBR1MCS-2 for protein pBBR1p264 production in Gluconobacter oxydans, Kmr pBBR1p264-GOX1253 pBBR1p264 with GOX1253 gene of G. oxydans 621H, Kmr pBBR1p264-GOX2071 pBBR1p264 with GOX2071 gene of G. oxydans 621H, Kmr a DSMZ, Deutsche Sammlung von Mikroorganismen und Zellkulturen, Braunschweig, Germany.

Source or reference DSMZa This study This study This study This study This study Invitrogen Invitrogen (24) Invitrogen Novagen This study This study (20) This study This study (23) This study This study

35

777

TABLE 2 Sequences of primers used in this study Primer

Sequence (5’→3’) and properties

GOX1253up.f

GGAATTCGATGTCAGCGTCACTAAAAGGC (EcoRI)

GOX1253up.r

ACGATGTCTTCAGCGCAGACGACCTGCTTG

GOX1253down.f CAAGCAGGTCGTCTGCGCTGAAGACATCGT GOX1253down.r TAAAAGCTTTCAGACGCTCTCGGCAACC (HindIII) GOX2071up.f

CCGGAATTCGATGCCGGAACCAGTCATGACCG (EcoRI)

GOX2071up.r

CACATGGCCCAGAAGTGACAGATCGAGGCT

GOX2071down.f AGCCTCGATCTGTCACTTCTGGGCCATGTG GOX2071down.r CCCAAGCTTTCAGCCCGTGTAAACAGCACC (HindIII) GOX1253C.f

GGGCATATGTCAGCGTCACTAAAAGGC (NdeI)

GOX1253C.r

TAAAAGCTTGACGCTCTCGGCAACCCAGTT (HindIII)

GOX2071C.f

AAGCATATGCCGGAACCAGTCATGA (NdeI)

GOX2071C.r

CAACTCGAGGCCCGTGTAAACAGCA (XhoI)

778 779

TABLE 3 Analysis on distribution of iLDHs in G. oxydans 621H using

780

ultracentrifugation Cell fraction

Specific activity (U/mg) D-iLDH

L-iLDH

Crude cell extract

0.15 ± 0.01

0.02 ± 0.00

Ultracentrifugation supernatant

0.02 ± 0.00

0.01 ± 0.00

Membrane fractions

0.40 ± 0.02

0.05 ± 0.00

781 782

36

783

TABLE 4 Location of GOX1253 and GOX2071 in G. oxydans 621H Specific activity of D-iLDH (U/mg) Strain G. oxydans ΔGOX1253

784

Ultracentrifugation

Membrane

supernatant

fractions

(0.50 ± 0.44) × 10-2

ND

a

G. oxydans ΔGOX2071

ND

0.41 ± 0.03

G. oxydans ΔGOX1253 ΔGOX2071

ND

ND

G. oxydans GOX2071 complement

(0.31 ± 0.10) × 10-2

ND

G. oxydans GOX1253 complement

0.58 ± 0.01

5.31 ± 0.15

a

ND, not detectable.

785 786

TABLE 5 Specific activities of GOX1253 and GOX2071 with different electron

787

acceptors Enzyme

Specific activity (U/mg)a DCPIP

MTT

K3[Fe(CN)6]

O2

GOX1253

54.23 ± 0.25

15.75 ± 0.83

6.01 ± 0.31

0.11 ± 0.03

GOX2071

0.61 ± 0.01

0.43 ± 0.01

3.95 ± 0.04

0.58 ± 0.02

788

a

789

Tris-HCl (pH 7.4), 20 mM D-lactate, appropriate amount of enzyme, and different

790

electron acceptors (0.05 mM DCPIP detected at 600 nm, 0.2 mM MTT at 578 nm, and 10

791

mM K3[Fe(CN)6] at 420 nm using a spectrophotometer, or 0.23 mM O2 at 900 rpm using

792

a Clark-type oxygen electrode).

The specific activities were examined in the reaction mixture containing 50 mM

37

793

TABLE 6 Identification of GOX2071 as a D-lactate oxidase System

Ingredients in reaction brothsa

Residual chemicals after reactionb

1.6 U/ml

20 mM

20 mM

100 U/ml

20 mM

D-Lactate

Pyruvate

GOX2071

D-lactate

pyruvate

catalase

H2O2

(mM)

(mM)

(mM)

1

+

+

–

–

–

10.14

2.03

7.59

2

+

+

–

+

–

11.49

8.53

0.22

3

+

–

+

–

–

NDc

20.35

ND

4

–

–

+

–

+

ND

0.46

19.55

5

–

–

+

+

+

ND

18.63

1.00

number

794

a

795

b

The data is the mean of three independent experiments.

796

c

ND, not detectable.

Acetate

Five aerobic reactions were conducted in the 50 mM Tris-HCl buffer (pH 7.4) at 30°C for 30 min.

797 798

TABLE 7 Apparent Km and Vmax of D-lactate and O2 for GOX2071 using a Clark-type oxygen electrode Km

Vmax

kcat

kcat/Km

(mM)

(U/mg)

(s-1)

(M-1 s-1)

D-Lactate

3.62 ± 0.53

0.52 ± 0.04

0.47 ± 0.04

129.74 ± 11.39

O2

0.16 ± 0.01

0.97 ± 0.03

0.87 ± 0.03

5423.59 ± 187.19

Substrate

799

38