1

Supplementary material

1. Acetoin and 2,3-BD are with various industrial applications

Acetoin and its derivatives have been applied in a variety of industries, for instance, cosmetics, pharmaceuticals and food flavoring.[1] As a flavoring and

fragrance agent, acetoin is generally recognized as safe (GRAS) by the Joint FAO/WHO Expert Committee on Food Additives (1998) and the US Food and Drug

Administration. Like FEMA No. 2008, acetoin is a common food additive.[2, 3] Acetoin is also employed in the synthesis of 2,3,5,6-tetramethylpyrazine(TTMP), a

biologically active ingredient in Chinese herbology that is routinely medicated in China.[4] Acetoin can be easily and efficiently oxidized from diacetyl, an acetoin

analogue with a strong buttery aroma.[5] Acetoin and its imine derivatives (acetol and acetoin) exert an extraordinarily strong stabilizing effect on alkoxides in the

titanium alkoxide-based sol–gel process.[6] Acetoin is also applied as a plant growth promoter in agriculture,[7] as an insect pheromone in pest control,[8] and as a

precursor in the fabrication of liquid crystal materials.[9]

2,3-BD is one important member of the C4-compound family with various industrial applications. The freezing point of (2S,3S)-2,3-BD is −60 °C, so it can be

utilized as an potentially valuable antifreeze agent.[10] With its heating value of 27.2 kJ/g, 2,3-BD is a promising fuel additive.[11] 2,3-BD can be converted to

methyl-ethyl-ketone, an industrial solvent,[12] or dehydrated to 2,3-butadiene, an important monomer of synthetic rubber,[13] or converted to diacetyl, a flavoring

agent in food products.[14] Elsewhere, 2,3-BD is variously applied in transport fuel production, printing ink manufacture, perfumes, explosives, and plasticizers.[15]

2

2. Metabolic pathway to acetoin and 2,3-BD: role of genes and their encoded enzymes

The biosynthesis of -acetolactate, acetoin and 2,3-BD mainly relies on Als, AldC, and Acr/BdhA, respectively. Their functions and roles in 2,3-BD synthesis have

been elucidated in several species.[16] For example, Lu et al. [17] observed the mutual interactions of these enzymes by mutated the K. pneumoniae KCTC2242::wabG

strain for single, double, and triple overproduction of the three enzymes. Although 2,3-BD yield was improved in all of the engineered strains, the highest yield of 2,3-

BD was obtained in the mutant budBA (overexpressing Als and AldC). The enzyme Als produces branched-chain amino acids that favor cell growth, AldC rapidly

enhances the transformation of acetolactate to acetoin in the presence of oxygen, and Acr/BdhA catalyzes the reversible conversion of acetoin to 2,3-BD and regulates

the intracellular NAD+/NADH balance.[17] Furthermore, Kim et al. [18] found that disruption of AldC can block the 2,3-BD pathway, increase amino acid (especially

valine) production, and decrease the CO2 emission during fermentation.

The metabolic step from acetoin to 2,3-BD is a primary reaction in this process. In many species (see supplementary material, Table S1), the reversible reaction in this

pathway is catalyzed by acetoin reductase (forward reaction) or 2,3-butanediol dehydrogenase (reverse reaction). These species include Bacillus cereus,[19] Bacillus

licheniformis,[20] other Bacillus sp. ,[21] Bacillus subtilis,[22, 23] Corynebacterium crenatum,[24] Clostridium beijerinckii,[25] Clostridium ljungdahlii,[26] Klebsiella

pneumoniae,[27-29] Paenibacillus polymyxa,[30-32] Rhodococcus erythropolis,[33, 34] Saccharomyces cerevisiae,[35, 36] and Serratia marcescens.[37] As shown in

3

Table S1, many Acr/BdhAs are NAD(H)-specific, but Acr/BdhAs from the Clostridium genus are NADP(H)-specific. All Acr/BdhAs exhibit a lower Km and higher

Km/Kcat for acetoin reduction than for 2,3-BD oxidation. The optimum pHs and temperatures of Acr/BdhAs, and the preferred pHs for reduction and oxidation,

largely differ among microorganisms. In Bacillus subtilis, Acr/BdhA is encoded by bdhA. However, Nicholson [23] detected a minimal amount of 2,3-BD in bdhA-

knockout mutants, suggesting that a second gene encodes minor BDH/AR activity. Yang et al. [38] found a similar gene (dar) in K. oxytoca with 53% identity to

budC, which is expressed along with budC. This gene encodes diacetyl/acetoin reductase (Dar), which plays an important role in 2,3-BD dissimilation in media

containing 2,3-BD alone. Furthermore, several researchers have proved the involvement of glycerol dehydrogenase in 2,3-BD production by K. pneumoniae [39] and

S. marcescens.[40]

BdhAs are stereospecific and divisible into three categories: (2R,3R)-BdhA, meso-BdhA, and (2S,3S)-BdhA, which are responsible for formation of corresponding

isomer of acetion and 2,3-BD.[41] Glycerol dehydrogenases (GDHs) also play major roles in the formation of 2,3-BD stereoisomers, and can convert (3R)-acetoin to

(2R,3R)-2,3-BD and diacetyl to meso-2,3-BD via (3S)-acetoin as an intermediate.[41, 42] In summary, the existence of multiple stereospecific dehydrogenases in

natural strains is thought to largely govern the mixed formation of acetoin and 2,3-BD stereoisomers.

4

Table S1 Characterization of 2,3-butanediol dehydrogenase/acetoin reductase from various strains

Source Gene Genbank

No. or GI

Optimal pH Optimal temperature

(°C) Coenzyme

specificity

Specificity

for 2,3-BD

isomers

Km (mM) kcat/Km (s-1·mM-1) References

Oxidation Reduction Oxidation Reduction BD AC DA BD AC DA

B. cereus YUF-

4

aacrII/

bdh

AB063194a ns 6.0-6.5 ns ns NADH/NAD+ R/meso 6.90R

10.35m

0.72R

0.98S

ns ns ns ns Hasaka etal.

(2001)

B.licheniformis budC 3100198 10.0 5.0 37 37 NADH/NAD+ S 7.25 0.47 72.4 81.5 432 16.9 Xu et al.

(2016)

B. poIymyxa

DSM365

D-bdh ns 9 7 60 60 NADH/NAD+ R/meso 3.3R

6.25m

0.53 87 ns ns ns Hohn-Bentz et

al.(1978)

Bacillus sp.

BRC1

bdh KF358987 9.0 6.0 50 50 NADH/NAD+ ns 6.01 0.12 0.13 ns ns ns Kang et al.

(2015)

B. subtilis JNA

3–10

bdhA ns 8.5 6.5 50 55 NADH/NAD+ ns 0.26 0.16 ns 5.40 2.13 ns Zhang et al.

(2014)

C. beijerinckii

NCIMB 8052

CBEI

1464

149902809 9.5 6.5 68 68 NADPH/NADP+ R ns 0.39 ns ns 214 ns Raedts et al.

(2014)

C. crenatum

SYPA5-5

butA KR611534 10.0 4.0 35 35 NADH/NAD+ ns 0.46 0.15 0.22 39.78 116.67 73.64 Zhao et al.

(2015)

C. ljungdahlii DSM 13528

CLJU

c23220

ns ns 8.0 ns 45 NADPH/NADP+ ns ns 0.14 ns ns 815.7 ns Tan et al.

(2015)

K. pneumoniae

ATCC 200721

ardII ABR76070 7 5 40 40 NADH/NAD+ S 5.51 0.58 3.80 ns ns ns Park et al.

(2014)

K. pneumoniae

XJ-Li

budC JN865245 9.0 8.0 35 35 NADH/NAD+ meso 13 0.65 ns ns ns ns Zhang et al.

(2012)

K. pneumoniae

LAM1063

Bdh ns 10-10.5 5-6 ns ns NADH/NAD+ meso 5.20 0.72 ns ns ns ns Ui et al.

(1997)

P. polymyxa

ZJ-9

bdh JN378394 8.0 6.0 80 30 NADH/NAD+ R/meso 7.67R

2.73m

0.20R

0.84S

ns ns ns ns Gao et al.

(2012)

P. polymyxa

ATCC 12321

bdh HQ730089 11.0 8.0 ns ns NADH/NAD+ R/meso 1.76R

5.62m

0.3 ns ns ns ns Yu et al.

(2011)

5

R. erythropolis

WZ010

rebdh KP868656 10.0 6.5 45 55 NADH/NAD+ R 0.58 ns 0.1 7.379 ns 61.1 Yu et al.

(2015)

R. erythropolis

WZ010

adr KC508606 9.5 7.0 25 30 NADH/NAD+ S 8.82 ns 0.44 0.061 ns 4.432 Wang et al.

(2014)

S. cerevisiae

BJ5459

bdh ns 8 7 ns ns NADH/NAD+ R 14 4.5 ns 93 364.2 ns Gonzalez et al.

(2001,2010)

S. marcescens

H30

budC AFH00999 8.0 5.0 40 40 NADH/NAD+ meso/S 4.1m

31.2S

0.97 3.3 1.51m

0.033S

20.3 3.48 Zhang et al.

(2013)

S. marcescens

B513

L-bdh ns 9 4.5 32-36 32-36 NADH/NAD+ meso 5 6.45 2.08 ns ns ns Hohn-Bentz et

al.(1978)

BD, 2,3-butanediol

AC, acetoin

DA, diacetyl

ns, not specified

a, DDBJ accession no.

6

Fig. S1 Stereoisomers of 2,3-BD and acetoin

Although different species produce different stereoisomeric forms, a mixture of two stereoisomers is usually produced.[1, 3, 14]

7

Fig. S2. Genetic organization of the 2,3-BD gene cluster in different species (not to scale).

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4613.

…………………

14

Figure 1

15

Figure 2