Production of Ethylene Glycol from Glycerol Using an in Vitro Enzymatic Cascade

catalysts

Communication Production of Ethylene Glycol from Glycerol Using an In Vitro Enzymatic Cascade

Kai Li, Weikang Sun, Wensi Meng, Jinxin Yan, Yipeng Zhang, Shiting Guo, Chuanjuan Lü *, Cuiqing Ma and Chao Gao

State Key Laboratory of Microbial Technology, Shandong University, Qingdao 266237, China; [email protected] (K.L.); [email protected] (W.S.); [email protected] (W.M.); [email protected] (J.Y.); [email protected] (Y.Z.); [email protected] (S.G.); [email protected] (C.M.); [email protected] (C.G.) * Correspondence: [email protected]; Tel.: +86-(532)-58631561

Abstract: Glycerol is a readily available and inexpensive substance that is mostly generated during biofuel production processes. In order to ensure the viability of the biofuel industry, it is essential to develop complementing technologies for the resource utilization of glycerol. Ethylene glycol is a two-carbon organic chemical with multiple applications and a huge market. In this study, an artiﬁcial enzymatic cascade comprised alditol oxidase, catalase, glyoxylate/hydroxypyruvate reductase, pyruvate decarboxylase and lactaldehyde:propanediol oxidoreductase was developed for the production of ethylene glycol from glycerol. The reduced nicotinamide adenine dinucleotide (NADH) generated during the dehydrogenation of the glycerol oxidation product D-glycerate can be as the reductant to support the ethylene glycol production. Using this in vitro synthetic system with

self-sufﬁcient NADH recycling, 7.64 ± 0.15 mM ethylene glycol was produced from 10 mM glycerol in 10 h, with a high yield of 0.515 ± 0.1 g/g. The in vitro enzymatic cascade is not only a promising

Citation: Li, K.; Sun, W.; Meng, W.; alternative for the generation of ethylene glycol but also a successful example of the value-added Yan, J.; Zhang, Y.; Guo, S.; Lü, C.; Ma, utilization of glycerol. C.; Gao, C. Production of Ethylene Glycol from Glycerol Using an In Keywords: glycerol; ethylene glycol; biocatalysis; in vitro enzymatic cascade Vitro Enzymatic Cascade. Catalysts 2021, 11, 214. https://doi.org/ 10.3390/catal11020214 1. Introduction Academic Editors: Peter Adewale The manufacturing of biofuels from reproducible feedstocks is increasing throughout and Michele C. Loewen the world [1–3]. The development of the biofuel industry has generated a large amount Received: 18 January 2021 of glycerol as an inevitable byproduct [4]. An excess of glycerol production in the biofuel Accepted: 2 February 2021 industry leads to a dramatic decrease of the price of glycerol, which has a negative impact Published: 5 February 2021 on the development of the biofuels industry [5]. Thus, the conversion of glycerol into value-added compounds through chemical or biotechnological routes is now urgently Publisher’s Note: MDPI stays neutral needed for the improvement of the viability of the biofuel industry. with regard to jurisdictional claims in published maps and institutional afﬁl- Ethylene glycol is a large-volume commodity that is widely used in many industrial iations. processes, such as glycolic acid production and polyethylene terephthalate synthesis [6]. It is predominantly produced from ethylene oxide through carbonation and subsequent hydrolyzation, or by the hydration of ethylene oxide [7,8]. Direct ethylene glycol production from biomass-derived carbohydrates through chemical catalysis has also been reported [9]. Importantly, ethylene glycol can be produced from glycerol through liquid-phase catalytic Copyright: © 2021 by the authors. hydrogenolysis [10,11]. However, this process usually needs high pressures (4.0–8.0 MPa), Licensee MDPI, Basel, Switzerland. high temperatures (453–473 K) and expensive noble metal catalysts (Pt, Ni, Ru, etc.). Several This article is an open access article distributed under the terms and metabolic pathways have been constructed for the biotechnological production of ethylene conditions of the Creative Commons glycol from renewable substrates such as D-glucose, D-xylose and L-arabinose [12–14]. Attribution (CC BY) license (https:// Nevertheless, the production of ethylene glycol from glycerol via a biotechnological route creativecommons.org/licenses/by/ has never been reported, until now. 4.0/).

Catalysts 2021, 11, 214. https://doi.org/10.3390/catal11020214 https://www.mdpi.com/journal/catalysts Catalysts 2021, 11, x FOR PEER REVIEW 2 of 9 Catalysts 2021, 11, 214 2 of 9

An in vitro synthetic system is emerging as a promising technique for the synthesis of highAn-invalue vitro productssynthetic from system cheap is emergingand renewable as a promising substrates technique [15,16]. This for thebiomanufactur- synthesis of high-valueing platform products leaves fromout the cheap use andof cells renewable, and can substrates thus exclude [15,16 cell]. This-associated biomanufacturing process bar- platform leaves out the use of cells, and can thus exclude cell-associated process barriers, riers, such as the transmembrane transport of substances, the toxicity of products, and such as the transmembrane transport of substances, the toxicity of products, and unde- undesired metabolism pathways. In addition, an in vitro synthetic system exclusively em- sired metabolism pathways. In addition, an in vitro synthetic system exclusively employs ploys purified enzymes, and makes the purification of the target products rather simple purified enzymes, and makes the purification of the target products rather simple to con- to conduct. Some in vitro synthetic systems have been constructed for the generation of duct. Some in vitro synthetic systems have been constructed for the generation of valuable valuable chemicals from renewable substrates, such as D-glucose, D-xylose and glycerol chemicals from renewable substrates, such as D-glucose, D-xylose and glycerol [17–19]. [17–19]. In this study, we have designed an artificial enzymic cascade for the conversion In this study, we have designed an artificial enzymic cascade for the conversion of glycerol of glycerol to ethylene glycol that only requires five enzymes. The artificial pathway is to ethylene glycol that only requires five enzymes. The artificial pathway is also com- also completely redox balanced through self-sufficient reduced nicotinamide adenine di- pletely redox balanced through self-sufficient reduced nicotinamide adenine dinucleotide nucleotide (NADH) recycling. (NADH) recycling.

2.2. ResultsResults andand DiscussionDiscussion 2.1.2.1. DesignDesign ofof thethe InInVitro Vitro Biosystem Biosystem for for Ethylene Ethylene Glycol Glycol Production Production Here,Here, an an artiﬁcial artificial enzymatic enzymatic reaction reaction cascade cascade was was designed designed for for the theconversion conversion of of glycerolglycerol intointo ethylene ethylene glycol glycol (Figure (Figure1 ).1). Glycerol Glycerol is is ﬁrst first oxidized oxidized to to DD-glycerate-glycerate via via DD-- glyceraldehydeglyceraldehyde byby alditol alditol oxidase oxidase (Aldo) (Aldo) catalyzed catalyzed two-step two-step oxidation. oxidation. The TheD D-Glycerate-Glycerate producedproduced fromfrom glycerolglycerol isis thenthen transformedtransformed into 3-hydroxypyruvate3-hydroxypyruvate by by oxidized oxidized nicotin- nicoti- namideamide adenine dinucleotide dinucleotide (NAD (NAD++)-dependent)-dependent glyoxylate/hydroxypyruvate glyoxylate/hydroxypyruvate reductase reductase (GRHPR).(GRHPR). Then,Then, 3-hydroxypyruvate3-hydroxypyruvate is is decarboxylated decarboxylated to to glycolaldehyde glycolaldehyde by by pyruvate pyruvate de- decarboxylasecarboxylase (P (PDC).DC). Finally, Finally, the the glycolaldehyde glycolaldehyde is isreduced reduced into into ethylene ethylene glycol glycol by by lactal- lac- taldehyde:propanedioldehyde:propanediol oxidoreductase oxidoreductase (FucO), (FucO), using using the the NADH NADH produced produced during during the the D- Dglycerate-glycerate dehydrogenation dehydrogenation as as the the cofactor. cofactor. The The H H2O2O2 generated2 generated during during the the oxidation oxidation of ofthe the glycerol glycerol is isdecomposed decomposed by by catalase catalase in inorder order to toavoid avoid the the spontaneous spontaneous oxidative oxidative of the of theintermediate intermediate products products [20]. [20 As]. As a aresult, result, one one mole mole of of glycerol glycerol can generate oneone molemole of of ethyleneethylene glycol. glycol.

FigureFigure 1. 1.Scheme Scheme of of ethylene ethylene glycol glycol production production from from glycerol glycerol based based on theon artiﬁcialthe artificialin vitro in vitroenzymatic enzy- cascade.matic cascade. The enzymes The enzymes are alditol are alditol oxidase oxidase (AldO) (AldO) from Streptomycesfrom Streptomyces coelicolor coelicolorA3(2), A3(2), catalase catalase from from Aspergillus niger, glyoxylate/hydroxypyruvate reductase (GRHPR) from Pyrococcus furiosus, Aspergillus niger, glyoxylate/hydroxypyruvate reductase (GRHPR) from Pyrococcus furiosus, pyruvate pyruvate decarboxylase (PDC) from Zymomonas mobile, and lactaldehyde:propanediol oxidoreduc- decarboxylase (PDC) from Zymomonas mobile, and lactaldehyde:propanediol oxidoreductase (FucO) tase (FucO) from Escherichia coli. from Escherichia coli.

2.2. Substrate Specificity of AldOmt 2.2. Substrate Specificity of AldOmt AldOAldO isis a a soluble soluble monomeric monomeric flavoprotein flavoprotein containing containing covalently-boundcovalently-bound FADFAD as as its its cofactorcofactor [ 21[21].]. ThisThis enzymeenzyme is is first first isolated isolated from fromStreptomyces Streptomyces coelicolor coelicolorA3(2) A3(2) and and identified identified asas an an oxidase oxidase primarily primarily active active with with alditols, alditols and, and as as producing producing aldoses aldoses as products.as products. Interest- Inter- ingly,estingly, AldO AldO exhibits exhibits regio- regio and- enantio-selectiveand enantio-selective oxidative oxidat activityive activity toward toward 1,2-diols 1,2- anddiols αand-hydroxy α-hydroxy carboxylic carboxylic acids butacids not but the notα-hydroxy the α-hydroxy aldehydes aldehydes which willwhich be will formed be formed as the endas the products end products [22]. AldO [22].mt AldOwithmt enhanced with enhanced oxidative oxidative activity activity towards towards glycerol glycerol was also was

CatalystsCatalysts 20212021,,11 11,, 214x FOR PEER REVIEW 33 ofof 99

constructedalso constructed through through directed directed evolution, evolution and this, and enzyme this enzyme has been has successfully been successfully applied ap- in Dplied-glycerate in D-glycerate production production from glycerol from glycerol [23]. [23]. The targettarget productproduct of of this this study, study, ethylene ethylene glycol, glycol, is theis the simplest simplest 1,2-diol, 1,2-diol and, and is also is also the possiblethe possible substrate substrate of AldO of AldOmt. Thus,mt. Thus, the the activity activity of AldO of AldOmt towardmt toward ethylene ethylene glycol glycol was was as- sayedassayed using using a Clark-type a Clark-type oxygen oxygen electrode, electrode as described, as described in a previous in a previous study [24study]. As [24]. shown As inshown Figure in2 a,Figure the AldO 2a, themt -dependentAldOmt-dependent oxygen consumptionoxygen consumption was rather was low rather with low the additionwith the ofaddition ethylene of glycol,ethylene while glycol, noticeable while oxygennoticeable consumption oxygen consumption was observed was when observed glycerol wh wasen addedglycerol (Figure was added2c). The (Figure estimated 2c). The speciﬁc estimated activities specific of activities AldO mt forof AldO glycerolmt for and glycerol ethylene and glycolethylene were glycol 0.53 were± 0.03 0.53 U/mg ± 0.03 and U/mg 0.018 and± 0.004 0.018 U/mg,± 0.004 respectivelyU/mg, respectively (Figure2 (Figureb ). The 2b). ox- idativeThe oxidative activities activities of AldO ofmt AldOtowardmt toward glycerol glycerol and ethylene and ethylene glycol glycol were alsowere determined also deter- bymined high-performance by high-performance liquid liquid chromatography chromatography (HPLC) (HPLC) analysis. analysis. As shown As shown in Figure in Figure2c,d , the2c,d depletion, the depletion of glycerol of glycerol could could be observed be observed obviously, obviously while, while very very little little consumption consumption of ethyleneof ethylene glycol glycol was was detected. detected.

Figure 2. Substrate specificityspecificity of AldOmtmt..( (aa)) The The d determinationetermination of of the the oxygen oxygen consumption consumption during during thethe AldOmt--catalyzedcatalyzed oxidation oxidation of of the the target target substrates. substrates. ( (bb)) The The s specificpecific activity activity of of AldO AldOmtmt towardtoward glycerol, DD-glyceraldehyde,-glyceraldehyde, glycolaldehyde,glycolaldehyde, and and ethylene ethylene glycol, glycol, respectively. respectively. The The error error bars bars indicate indicate the standard deviations from three parallel replicates. (c,d) HPLC analysis of AldOmt-cata- the standard deviations from three parallel replicates. (c,d) HPLC analysis of AldOmt-catalyzed lyzed glycerol (c) or ethylene glycol (d) oxidation. The reactions were carried out in a reaction mix- glycerol (c) or ethylene glycol (d) oxidation. The reactions were carried out in a reaction mixture con- ture containing 50 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES)-NaOH buffer taining 50 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES)-NaOH buffer (pH 7.4), (pH 7.4), 10 mM glycerol or 10 mM ethylene glycol and 0.2 mg/mL AldO◦ mt, at 30 °C, with a shak- 10ing mM speed glycerol of 150 or rpm. 10 mM ethylene glycol and 0.2 mg/mL AldOmt, at 30 C, with a shaking speed of 150 rpm. AldO oxidizes glycerol into D-glycerate in a two-step reaction via D-glyceraldehyde AldO oxidizes glycerol into D-glycerate in a two-step reaction via D-glyceraldehyde [23]. [23]. Glycolaldehyde, the direct precursor of ethylene glycol in the designed enzymatic Glycolaldehyde, the direct precursor of ethylene glycol in the designed enzymatic reaction reaction cascade, is the analogue of D-glyceraldehyde, and might be oxidized by AldOmt. cascade, is the analogue of D-glyceraldehyde, and might be oxidized by AldOmt. Thus, Thus, the activities of AldOmt toward glycolaldehyde and D-glyceraldehyde were also as- the activities of AldO toward glycolaldehyde and D-glyceraldehyde were also assayed. sayed. As shown in Figuremt 2a,b, a very much lower oxygen consumption rate was ob- As shown in Figure2a,b, a very much lower oxygen consumption rate was observed when served when glycolaldehyde, but not D-glyceraldehyde, was added to the reaction mix- glycolaldehyde, but not D-glyceraldehyde, was added to the reaction mixture, indicating ture, indicating that AldOmt exhibits rather low oxidative activity toward glycolaldehyde. that AldOmt exhibits rather low oxidative activity toward glycolaldehyde. The result of The result of the HPLC analysis further confirmed that AldOmt can hardly catalyze the the HPLC analysis further confirmed that AldOmt can hardly catalyze the oxidation of glycolaldehydeoxidation of glycolaldehyde (Figure S1). (Figure S1).

Catalysts 2021, 11, x FOR PEER REVIEW 4 of 9 Catalysts 2021, 11, 214 4 of 9

Based on the data mentioned above, AldOmt can efficiently oxidize glycerol into D- glycerateBased, onbut thewill data barely mentioned consume above,the glycolalde AldOmthydecan and efﬁciently ethylene oxidize glycol glycerolin the reaction into D-glycerate,system. Thus, but AldO will barelymt is a consumeproper catalyst the glycolaldehyde in the designed and enzymatic ethylene glycol reaction inthe cascade reaction, and system.it exhibits Thus, little AldO promiscuousmt is a proper activity catalyst affecting in the designedthe production enzymatic of ethylene reaction glycol. cascade, and it exhibits little promiscuous activity affecting the production of ethylene glycol. 2.3. 3-Hydroxypyruvate Decarboxylation Catalyzed by PDC 2.3. 3-Hydroxypyruvate Decarboxylation Catalyzed by PDC The GRHPR of Pyrococcus furiosus can catalyze the NAD+-dependent dehydrogena- The GRHPR of Pyrococcus furiosus can catalyze the NAD+-dependent dehydrogenation tion of D-glycerate to produce 3-hydroxypyruvate [18,25]. However, the standard Gibbs of D-glycerate to produceθ 3-hydroxypyruvate [18,25]. However, the standard Gibbs energy energy changeθ (ΔrG ) of D-glycerate dehydrogenation is estimated to be 20.26 KJ/mol, in- change (∆rG ) of D-glycerate dehydrogenation is estimated to be 20.26 KJ/mol, indicating dicating that this reaction is not thermodynamically favorable. In order to support the that this reaction is not thermodynamically favorable. In order to support the GRHPR- GRHPR-catalyzed, thermodynamically unfavorable D-glycerate dehydrogenation, the in- catalyzed, thermodynamically unfavorable D-glycerate dehydrogenation, the integration of tegration of thermodynamically favorable reactions with 3-hydroxypyruvate or NADH thermodynamically favorable reactions with 3-hydroxypyruvate or NADH as the substrates as the substrates is needed. is needed. The PDC of Zymomonas mobilis can catalyze the decarboxylation of pyruvate to pro- The PDC of Zymomonas mobilis can catalyze the decarboxylation of pyruvate to produce carbon dioxide and acetaldehyde [26]. In this study, the activity of PDC toward 3- duce carbon dioxide and acetaldehyde [26]. In this study, the activity of PDC toward 3- hydroxypyruvate decarboxylation, a thermodynamically favorable reaction (ΔrGθ = hydroxypyruvate decarboxylation, a thermodynamically favorable reaction −55.23 KJ/mol), was also assayed. As shown in Figure S2, PDC also exhibited decarboxy- (∆rGθ = −55.23 KJ/mol), was also assayed. As shown in Figure S2, PDC also exhibited decarboxylationlation activity to activity catalyze to catalyze3-hydroxypyruvate 3-hydroxypyruvate into glycolaldehyde. into glycolaldehyde. About 8 About mM glycolal- 8 mM glycolaldehydedehyde was produced was produced from 10 from mM 10 3- mMhydroxypyruvate 3-hydroxypyruvate in 4 h inby 4 10 h byμg/mL 10 µ g/mLPDC. PDC.

2.4.2.4. Substrate Substrate Speciﬁcity Specificity of of FucO FucO TheThe NADH-dependent NADH-dependent reduction reduction of of glycolaldehyde glycolaldehyde can can regenerate regenerate the the NAD NAD+ +re- re- quiredquired for forD-glycerate D-glycerate dehydrogenation, dehydrogenation and, and can can support support the the production production of ethylene of ethylene glycol. gly- Thecol. lactaldehyde:propanediol The lactaldehyde:propanediol oxidoreductase oxidoreductase of Escherichia of Escherichia coli (FucO) coli (FucO) was reportedwas reported to reduceto reduce glycolaldehyde glycolaldehyde [27, 28[27,28].]. This This enzyme enzyme has has been been overexpressed overexpressed in inE. E coli. colifor forin in vivo vivo ethyleneethylene glycol glycol production production via via a synthetica synthetic pathway pathway [29 [29].]. In In this this study, study, the the activity activity of of FucO FucO usingusing glycolaldehyde glycolaldehyde and and NADH NADH as theas substratesthe substrates was alsowas assayedalso assayed (Figure (Figure3a,b ). 3a The,b). spe- The ciﬁcspecific activity activity of FucO of towardFucO toward glycolaldehyde glycolaldehyde reduction reduction was calculated was calculated to be 30.9 to± be1.2 30.9 U/mg ± 1.2 (FigureU/mg3 (Figureb ). The 3b). HPLC The analysis HPLC analysis results showed results showed that FucO that rapidly FucO reducesrapidly glycolaldehydereduces glycolal- intodehyde ethylene into glycolethylene (Figure glycol3c). (Figure 3c).

FigureFigure 3. 3.Substrate Substrate specificity specificity of FucO.of FucO. (a) Time(a) Time course course of reactions of reactions of FucO of FucO with glycerol,with glycerol,D-glyceraldehyde, D-glyceraldehyde, glycolaldehyde, glycolalde- orhyde ethylene, or glycolethylene as theglycol substrate as the and substrate NADH and as the NADH cofactor. as (theb) Specific cofactor. activity (b) Specific of FucO activity toward of glycerol, FucO towardD-glyceraldehyde, glycerol, D- glyceraldehyde, glycolaldehyde and ethylene glycol, respectively. The error bars indicate the standard deviations from glycolaldehyde and ethylene glycol, respectively. The error bars indicate the standard deviations from three parallel three parallel replicates. (c) HPLC analysis of the FucO-catalyzed glycolaldehyde reduction. The reaction was carried out replicates. (c) HPLC analysis of the FucO-catalyzed glycolaldehyde reduction. The reaction was carried out in a reaction in a reaction mixture containing 50 mM HEPES-NaOH buffer (pH 7.4), 10 mM glycolaldehyde, 10 mM NADH and 0.5 mixturemg/mL containing FucO, at 30 50 °C mM, with HEPES-NaOH a shaking speed buffer of (pH 150 7.4), rpm. 10 mM glycolaldehyde, 10 mM NADH and 0.5 mg/mL FucO, at 30 ◦C, with a shaking speed of 150 rpm. The activities of FucO toward other reactants in the in vitro system including eth- The activities of FucO toward other reactants in the in vitro system including ethylene ylene glycol, glycerol and D-glyceraldehyde were also assayed. As shown in Figure 3a,b, glycol, glycerol and D-glyceraldehyde were also assayed. As shown in Figure3a,b, all of all of these reactant especially D-glyceraldehyde, the analogue glycolaldehyde, cannot be these reactant especially D-glyceraldehyde, the analogue glycolaldehyde, cannot be reduced reduced by FucO. Thus, the NADH generated during D-glycerate oxidation can only be by FucO. Thus, the NADH generated during D-glycerate oxidation can only be consumed consumed for glycolaldehyde reduction. This property of FucO ensured the self-sufficient for glycolaldehyde reduction. This property of FucO ensured the self-sufficient NADH

Catalysts 2021, 11, x FOR PEER REVIEW 5 of 9

Catalysts 2021, 11, 214 5 of 9

NADH recycling of the in vitro system, and avoided the requirement for exogenous reducing agent addition. recycling of the in vitro system, and avoided the requirement for exogenous reducing 2.5.agent Production addition. of Ethylene Glycol from Glycerol In Vitro 2.5.AldO Productionmt, GRHPR, of Ethylene PDC Glycol and FucO from Glycerolwere overexpressed, In Vitro purified (Figure S3), and coupled with commercial catalase from A. niger (Sigma-Aldrich, St. Louis, MO, USA) for the AldO , GRHPR, PDC and FucO were overexpressed, purified (Figure S3), and production mtof ethylene glycol from glycerol. As shown in Figure 4, 7.64 ± 0.15 mM eth- coupled with commercial catalase from A. niger (Sigma-Aldrich, St. Louis, MO, USA) for ylene glycol was generated from 10 mM glycerol in 10 h. Some chemists have also at- the production of ethylene glycol from glycerol. As shown in Figure4, 7.64 ± 0.15 mM tempted to produce ethylene glycol from glycerol by hydrogenolysis. For example, Feng ethylene glycol was generated from 10 mM glycerol in 10 h. Some chemists have also and his colleagues studied the conversion of glycerol to ethylene glycol over Ru-Co/ZrO2 attempted to produce ethylene glycol from glycerol by hydrogenolysis. For example, Feng catalysts [30]. However, this catalyst exhibited higher selectivity toward 1,2-propanediol and his colleagues studied the conversion of glycerol to ethylene glycol over Ru-Co/ZrO2 generation than ethylene glycol production. The yield of ethylene glycol was only 0.30 g/g catalysts [30]. However, this catalyst exhibited higher selectivity toward 1,2-propanediol glycerol.generation Ueda than and ethylene his colleagues glycol production. reported the The co yieldnversion of ethylene of glycerol glycol to was ethylene only 0.30 glycol g/g overglycerol. Pt-modified Ueda and Ni catalysts his colleagues under reported the conditions the conversion of 453 K ofand glycerol 8.0 MPa to [10]. ethylene The glycolyield ofover ethylene Pt-modified glycol was Ni catalysts0.479 g/g, under which the was conditions lower than of 453that K of and a biocatalytic 8.0 MPa [10 process]. The yielduti- lizingof ethylene the artificially glycol wasdesigned 0.479 enzyma g/g, whichtic cascade was lower (0.515 than ± 0.1 that g/g) of under a biocatalytic modest reaction process conditionutilizings the (303 artificially K, 0.1 MPa). designed enzymatic cascade (0.515 ± 0.1 g/g) under modest reaction conditions (303 K, 0.1 MPa).

FigureFigure 4. 4.ConversionConversion of ofglycerol glycerol into into ethylene ethylene glycol glycol by bythe theartificial artiﬁcial enzymatic enzymatic reaction reaction cascade. cascade. TheThe reaction reaction was was carried carried out out in in a areaction reaction mixture mixture containing containing 50 50 mM mM HEPES HEPES-NaOH-NaOH buffer buffer (pH (pH 7.4), + + 7.4),10 mM10 mM glycerol, glycerol, 1 mM 1 mM NAD NAD, 2, mM2 mM MgSO MgSO4,4 0.25, 0.25 mM mM thiamine thiamine pyrophosphate pyrophosphate (TPP), (TPP), 1 1 mg/mL mg/mLAldOmt AldO, 10 mg/mLmt, 10 mg/mL GRHPR, GRHPR, 5.4 mg/mL 5.4 mg/mL PDC, 13PDC, mg/mL 13 mg/mL FucO andFucO 500 and U/mL 500 U/mL catalase. catalase. The reaction The reactionwas conducted was conducted at 30 ◦ atC 30 and °C a and shaking a shaking speed speed of 150 of 150 rpm. rpm. The The error error bars bars indicate indicate the the standard stand- arddeviations deviations from from three three parallel parallel replicates. replicates.

SomeSome biotechnological biotechnological routes routes have have been been developed developed for for the the production production of of ethylene ethylene glycolglycol from from biomass biomass-derived-derived carbohydrates carbohydrates [12 [–1214,19,29,31–14,19,29,–3135]–35 (Table] (Table 1).1 For). For example, example, D- glucoseD-glucose was was used used as a as substrate a substrate to produce to produce ethylene ethylene glycol glycol via via the the extension extension of of the the serine serine biobiosynthesissynthesis pathway. pathway. The The yields yields of ofethylene ethylene glycol glycol from from D-glucoseD-glucose were were only only 0.09 0.09 g/g g/gby engineeredby engineered CorynebacteriumCorynebacterium glutamicum glutamicum and 0.14and g/g 0.14 by g/g engineered by engineered E. coli E.[14,31]. coli [14 Glycol,31]. Gly-al- dehyde,colaldehyde, the direct the precursor direct precursor of ethylene of ethylene glycol, can glycol, be produced can be produced from D-xylose from Dor-xylose xylonic or acidxylonic through acid the through Dahms the pathway Dahms [12,29,32] pathway. [Due12,29 to,32 the]. Duecarbon to theloss carboninduced loss by inducedthe copro- by ductionthe coproduction of pyruvate, of the pyruvate, theoretical the theoreticalyield of ethylene yield of glycol ethylene from glycol D-xylose from waDs-xylose 0.413 g/g was. Liu0.413 and g/g. his colleagues Liu and his disrupted colleagues the D disrupted-xylose isomerisation the D-xylose pathway isomerisation and introduced pathway andyjr Dahmsintroduced pathway Dahms in E. pathway coli, and in obtainedE. coli, and a yield obtained of 0.29 a yield g/g ofethylene 0.29 g/g glycol ethylene from glycol D-xylose from [12].D-xylose A higher [12]. yield A higher of 0.40 yield g/g of 0.40ethylene g/g ethylene glycol from glycol D from-xyloseD-xylose was obtained was obtained by further by fur- improvingther improving the redox the redoxbalance balance of the ofDahms the Dahms pathway, pathway, overexpressing overexpressing FucO FucOand YjhG, and YjhG,and deletingand deleting the by the-products by-products encoding encoding genes genes aldA aldAand arcAand arcA[29].[ Recently,29]. Recently, a cell a- cell-freefree bioreac- biore- tionaction scheme scheme was was also also reported reported to togenerate generate ethylene ethylene glycol glycol from from D-Dxylose-xylose,, with with a ayield yield of of 0.227 g/g [19]. In this study, we designed an artiﬁcial enzymatic cascade to produce ethylene glycol from glycerol with only one carbon atom loss. A high yield of 0.515 ± 0.1 g/g ethylene glycol from glycerol was acquired using the in vitro synthetic system.

Catalysts 2021, 11, 214 6 of 9

Table 1. Yield of ethylene glycol production from various substrates by metabolically-engineered strains or an in vitro biosystem.

Strain or Enzymes Substrate Ethylene Glycol Yield (g/g) Reference E. coli BW25113(DE3) D-Xylose 0.29 [12] ∆xylA∆aldA/pACYC-xdh-yqhD E. coli MG1655 ∆xylB/pEXT20-khkC-aldoB D-Xylose 0.19 [13] E. coli BL21(DE3) ∆arcA∆aldA/pETDuet1-yjhH-xdh- D-Xylose 0.40 [29] xylC/pACYCDuet1-fucO-yjhG E. coli MG1655(DE3) ∆endA∆recA D-Xylose 0.35 [33] ∆xylB∆aldA/p10_T5-Pc.dte-fucA-fucO-fucK E. coli W3110 ∆lacI/pTacxylBC-yqhD-P1-anti-xylB D-Xylose 0.36 [34] E. coli W3110 ∆xylAB∆aldA∆yjgB∆yqhD/pKMX & D-Xylose 0.39 [35] pTrcHis2A-yqhD Enterobacter cloacae S1 Xylonic acid 0.288 [32] E. coli MG1655(DE3) ∆endA∆recA∆xylB∆araB∆aldA/p5_T7-Pc.dte-rhaB- L-Arabinose 0.38 [33] rhaD-fucO E. coli MG1655(DE3) ∆ald∆AeutBC/p10_T7-serA:317-serB-serC-fucO & D-Glucose 0.14 [31] pCDFDuet_T7-At.sdc.t+T7-aao C. glutamicum PABS1/pEC-P1-GsLdh-PpMdlc-yqhD D-Glucose 0.09 [14] D-Xylose dehydrogenase, xylonolactonase, xylonate dehydratase, 2-keto-3-deoxy-D-xylonate aldolase, D-Xylose 0.227 [19] lactaldehyde reductase, α-acetolactate synthase, α-acetolactate decarboxylase AldOmt, catalase, GRHPR, PDC, FucO Glycerol 0.515 This study

The in vitro synthetic system has appealing advantages, such as a high product yield, easy product separation, and simple process control [16]. It has made great progress in recent years. For example, the production of inositol from starch using the in vitro synthetic system has been implemented on an industrial scale [36]. In this study, we designed an enzymatic cascade for the production of ethylene glycol from glycerol. Although a higher yield of ethylene glycol was obtained using the artificially designed enzymatic cascade, the final concentration and productivity of ethylene glycol cannot meet the requirements for industrial application. The low activity of GRHPR may be the key factor restricting the efficiency of the in vitro enzymatic cascade. Systematic screening or the directed evolution of a new biocatalyst with higher activity toward the dehydrogenation of D-glycerate to 3-hydroxypyruvate will be necessary to further improve the production of ethylene glycol. The optimization of the reaction system may also improve the efficiency of the enzymatic cascade, and this deserves an intensive study [37].

3. Materials and Methods Materials: reduced/oxidized nicotinamide adenine dinucleotide (NADH/NAD+), isopropyl-β-D-1-thiogalactopyranoside (IPTG), thiamine pyrophosphate (TPP), 3-hydroxypyruvate, D-glycerate and glycolaldehyde were purchased from Sigma-Aldrich (St. Louis, MO, USA). Restriction endonuclease and T4 DNA ligase were purchased from Thermo Scientiﬁc (Waltham, MA, USA). FastPfu DNA polymerase was purchased from TransGen Biotech Ltd. (Beijing, China). The other chemicals used in this study were analytical pure grade and commercially available. The cloning and expression of fucO: the strains, plasmids and primers used in this study are listed in Supplementary Materials Table S1. E. coli DH5α was used for the plasmid construction, and E. coli BL21(DE3) was used for the overexpression of the recombinant proteins. The encoding gene of FucO was ampliﬁed from the genomic DNA of E. coli K12 with the primers fucO-f/fucO-r (Supplementary Table S1), digested with BamHI/HindIII, and cloned into pET28a in order to construct pET28a-FucO. The plasmid pET28a-FucO was then transferred into E. coli BL21(DE3) to obtain E. coli BL21(DE3)/pET28a-FucO. All of the E. coli strains were cultivated in Luria-Bertani (LB) medium at 37 ◦C and 180 rpm or 16 ◦C Catalysts 2021, 11, 214 7 of 9

and 160 rpm. If necessary, ampicillin (at a final concentration of 100 µg/mL) or kanamycin (at a final concentration of 50 µg/mL) was added into the medium. Purification of the enzymes: the catalase from A. niger used in this study was purchased from Sigma-Aldrich (St. Louis, MO, USA). The AldOmt, GRHPR and PDC were purified as described by Li and his colleagues [20]. E. coli BL21(DE3)/pET28a-FucO was grown at 37 ◦C and 180 rpm in LB medium with 100 µg/mL kanamycin to an optical density of 0.5 at 620 nm. Then, IPTG was added at a final concentration of 1.0 mM in order to induce the expression of FucO. After cultivation for another 4 h at 37 ◦C, the cells were harvested and washed twice with 20 mM phosphate solution buffer (PBS, pH 7.4) by centrifugation at 6000 rpm for 10 min; the cells were subsequently suspended in binding buffer (20 mM phosphate buffer, 500 mM NaCl, and 20 mM imidazole, pH 7.4) and lysed using a continuous high-pressure cell disrupter working at 4 ◦C and 900 bar. The cell lysate was centrifugated at 12,000 rpm for 1 h, and the supernatant then was loaded onto a 5 mL HisTrap HP column (GE Healthcare, Sweden). The target proteins were eluted with a gradient ratio of elution buffer (20 mM phosphate buffer, 500 mM NaCl, and 500 mM imidazole, pH 7.4). The purified proteins including AldOmt, GRHPR, PDC and FucO were subjected to sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE), and the results are shown in Supplementary Figure S3. Ethylene glycol production: the production of ethylene glycol from glycerol was performed in a 5 mL reaction system containing 1 mg/mL AldOmt, 500 U/mL catalase, 10 mg/mL GRHPR, 5.4 mg/mL PDC, 13.0 mg/mL FucO, 10 mM glycerol, 1 mM NAD+, ◦ 0.25 mM TPP, 2 mM MgSO4, and 50 mM HEPES-NaOH (pH 7.4) at 30 C and 150 rpm. Samples (0.2 mL) were withdrawn every 2 h and subjected to analysis by HPLC. Analytical methods: the concentrations of the purified proteins were measured by the use of the Bradford protein assay (Bio-Rad, Hercules, CA, USA). The activity of the FucO was assayed by recording the absorption of NADH at 340 nm using a UV–visible spectrophotometer (Ultrospec 2100 pro; Amersham Biosciences, Little Chalfont, UK) [20]. All of the reactions were carried out in a 1.0 mL quartz cuvette at 30 ◦C, and HEPES- NaOH buffer (50 mM, pH 7.4) with 0.2 mM NADH and 2.5 mM substrate (glycerol, D- glyceraldehyde, glycolaldehyde or ethylene glycol). One unit of FucO activity was defined as the amount of protein catalyzing the consumption of one µmol of NADH per minute. The oxidase activities of AldOmt toward glycerol, D-glyceraldehyde, glycolaldehyde and ethylene glycol were assayed by measuring the rate of oxygen consumption. The reaction mixture contained 10 mM substrate, 50 mM HEPES-NaOH buffer (pH 7.4), and 0.2 mg/mL AldOmt. The oxygen consumption in the reaction system was monitored using a Clark-type oxygen electrode (Oxytherm System; Hansatech Instruments Ltd., Pentney, UK). One unit of AldOmt activity was defined as the amount of protein catalyzing the consumption of one µmol O2 per minute. The concentrations of glycerol, glyceraldehyde, D-glycerate and 3-hydroxypyruvate were analyzed using an HPLC system (Agilent 1100 series, Hewlett-Packard, Palo Alto, CA, USA) equipped with an anion exchange column (Aminex HPX-87H, 300 × 7.8 mm; Bio-Rad, USA) and a refractive index detector [20]. The mobile phase was 5.0 mM H2SO4 at a flow rate of 0.4 mL/min and at a temperature of 55 ◦C. The concentrations of the analytes were calculated by converting the peak areas detected by HPLC using external calibration curves.

4. Conclusions In conclusion, we designed an artiﬁcial in vitro biosystem involving ﬁve enzymes for the production of ethylene glycol from glycerol. Ethylene glycol was produced from glycerol with a yield of 0.515 ± 0.1 g/g, which is higher than any reported chemical or biotechnological routes to date. The method presented in this work would not only provide a promising process for ethylene glycol production, but would also expand the utilization of the glycerol produced by the biofuel industry. Catalysts 2021, 11, 214 8 of 9

Supplementary Materials: The following are available online at https://www.mdpi.com/2073 -4344/11/2/214/s1. Figure S1: HPLC analysis of AldOmt-catalyzed D-glyceraldehyde (a) and glycolaldehyde (b) oxidation. The reactions were carried out in a reaction mixture containing 50 mM HEPES-NaOH buffer (pH 7.4), 0.2 mg/mL AldOmt, 10 mM D-glyceraldehyde or 10 mM glycolaldehyde, at 30 ◦C and 150 rpm. Figure S2: HPLC analysis of PDC catalyzed 3-hydroxypyruvate decarboxylation. The reaction was carried out in 50 mM HEPES-NaOH buffer (pH 7.4) containing 2 ◦ mM MgSO4, 0.25 mM TPP, 10 mM 3-hydroxypyruvate and 10 µg/mL PDC, at 30 C and 150 rpm. Figure S3: SDS-PAGE results of the purified enzymes. M: Marker; 1: AldOmt; 2: GRHPR; 3: PDC; 4: FucO. Table S1: Strains, plasmids and primers used in this study. Author Contributions: Conceptualization, methodology, formal analysis, investigation, data cura- tion, K.L. and C.L.; software, validation, Y.Z. and S.G.; resources, K.L., J.Y. and W.S.; writing—original draft preparation, K.L., W.M. and C.G.; writing—review and editing, supervision, project administra- tion, C.L. and C.M.; funding acquisition, C.G., C.L. and C.M. All authors have read and agreed to the published version of the manuscript. Funding: This research was funded by the National Key R&D Program of China, grant number 2019YFA0904900, 2019YFA0904800; Shandong Provincial Funds for Distinguished Young Scien- tists, grant number JQ 201806; Natural Science Foundation of Shandong Provincial, grant number ZR2018PC008, ZR2020MC005; Tianjin Synthetic Biotechnology Innovation Capacity Improvement Project, grant number TSBICIP-KJGG-005; and the China Postdoctoral Science Foundation, grant number 62450078311268. Acknowledgments: The authors thank Caiyun Sun from Core Facilities for Life and Environmental Sciences (State Key Laboratory of Microbial Technology, Shandong University) for assistance in the HPLC analysis. Conflicts of Interest: The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

References 1. Mahmood, W.M.A.; Theodoropoulos, C.; Gonzalez-Miquel, M. Enhanced microalgal lipid extraction using bio-based solvents for sustainable biofuel production. Green Chem. 2017, 19, 5723–5733. [CrossRef] 2. Hill, J.; Nelson, E.; Tilman, D.; Polasky, S.; Tiffany, D. Environmental, economic, and energetic costs and benefits of biodiesel and ethanol biofuels. Proc. Natl. Acad. Sci. USA 2006, 103, 11206–11210. [CrossRef][PubMed] 3. Heeres, A.S.; Picone, C.S.; van der Wielen, L.A.; Cunha, R.L.; Cuellar, M.C. Microbial advanced biofuels production: Over-coming emulsification challenges for large-scale operation. Trends Biotechnol. 2014, 32, 221–229. [CrossRef] 4. Tan, H.W.; Abdul Aziz, A.; Aroua, M. Glycerol production and its applications as a raw material: A review. Renew. Sustain. Energy Rev. 2013, 27, 118–127. [CrossRef] 5. Lu, X.; Vora, H.; Khosla, C. Overproduction of free fatty acids in E. coli: Implications for biodiesel production. Metab. Eng. 2008, 10, 333–339. [CrossRef][PubMed] 6. Yue, H.; Zhao, Y.; Ma, X.; Gong, J. Ethylene glycol: Properties, synthesis, and applications. Chem. Soc. Rev. 2012, 41, 4218–4244. [CrossRef] 7. Kawabe, K. Development of highly selective process for mono-ethylene glycol production from ethylene oxide via ethylene carbonate using phosphonium salt catalyst. Catal. Surv. Asia 2010, 14, 111–115. [CrossRef] 8. Li, Y.; Yue, B.; Yan, S.; Yang, W.; Xie, Z.; Chen, Q.; He, H. Preparation of ethylene glycol via catalytic hydration with highly efficient supported niobia catalyst. Catal. Lett. 2004, 95, 163–166. [CrossRef] 9. Salusjärvi, L.; Havukainen, S.; Koivistoinen, O.; Toivari, M. Biotechnological production of glycolic acid and ethylene glycol: Current state and perspectives. Appl. Microbiol. Biotechnol. 2019, 103, 2525–2535. [CrossRef] 10. Ueda, N.; Nakagawa, Y.; Tomishige, K. Conversion of glycerol to ethylene glycol over Pt-modified Ni catalyst. Chem. Lett. 2010, 39, 506–507. [CrossRef] 11. Yin, A.-Y.; Guo, X.-Y.; Dai, W.-L.; Fan, K.-N. The synthesis of propylene glycol and ethylene glycol from glycerol using Raney Ni as a versatile catalyst. Green Chem. 2009, 11, 1514–1516. [CrossRef] 12. Liu, H.; Ramos, K.R.M.; Valdehuesa, K.N.G.; Nisola, G.M.; Lee, W.-K.; Chung, W.-J. Biosynthesis of ethylene glycol in Escherichia coli. Appl. Microbiol. Biotechnol. 2012, 97, 3409–3417. [CrossRef] 13. Cam, Y.; Alkim, C.; Trichez, D.; Trebosc, V.; Vax, A.; Bartolo, F.; Besse, P.; François, J.-M.; Walther, T. Engineering of a synthetic metabolic pathway for the assimilation of (D)-Xylose into value-added chemicals. ACS Synth. Biol. 2015, 5, 607–618. [CrossRef] 14. Chen, Z.; Huang, J.; Wu, Y.; Liu, D. Metabolic engineering of Corynebacterium glutamicum for the de novo production of ethylene glycol from glucose. Metab. Eng. 2016, 33, 12–18. [CrossRef] Catalysts 2021, 11, 214 9 of 9

15. Rollin, J.A.; Tam, T.K.; Zhang, Y.-H.P. New biotechnology paradigm: Cell-free biosystems for biomanufacturing. Green Chem. 2013, 15, 1708–1719. [CrossRef] 16. Shi, T.; Han, P.; You, C.; Zhang, Y.P.J. An in vitro synthetic biology platform for emerging industrial biomanufacturing: Bot-tom-up pathway design. Synth. Syst. Biotechnol. 2018, 3, 186–195. [CrossRef] 17. Gao, C.; Li, Z.; Zhang, L.; Wang, C.; Li, K.; Ma, C.; Xu, P. An artificial enzymatic reaction cascade for a cell-free bio-system based on glycerol. Green Chem. 2015, 17, 804–807. [CrossRef] 18. Li, Z.; Yan, J.; Sun, J.; Xu, P.; Ma, C.; Gao, C. Production of value-added chemicals from glycerol using in vitro enzymatic cascades. Commun. Chem. 2018, 1, 1–7. [CrossRef] 19. Jia, X.; Kelly, M.R.; Han, Y. Simultaneous biosynthesis of (R)-acetoin and ethylene glycol from D-xylose through in vitro met-abolic engineering. Metab. Eng. Commun. 2018, 7, e00074. [CrossRef] 20. Li, Z.; Zhang, M.; Jiang, T.; Sheng, B.; Ma, C.; Xu, P.; Gao, C. Enzymatic cascades for efficient biotransformation of racemic lactate derived from corn steep water. ACS Sustain. Chem. Eng. 2017, 5, 3456–3464. [CrossRef] 21. Heuts, D.P.H.M.; Van Hellemond, E.W.; Janssen, D.B.; Fraaije, M.W. Discovery, characterization, and kinetic analysis of an alditol oxidase from Streptomyces coelicolor. J. Biol. Chem. 2007, 282, 20283–20291. [CrossRef] 22. Forneris, F.; Heuts, D.P.H.M.; Delvecchio, M.; Rovida, S.; Fraaije, M.W.; Mattevi, A. Structural analysis of the catalytic mechanism and stereoselectivity in Streptomyces coelicolor alditol oxidase. Biochemistry 2008, 47, 978–985. [CrossRef] 23. Gerstenbruch, S.; Wulf, H.; Mußmann, N.; O’Connell, T.; Maurer, K.-H.; Bornscheuer, U.T.; Mussmann, N. Asymmetric synthesis of D-glyceric acid by an alditol oxidase and directed evolution for enhanced oxidative activity towards glycerol. Appl. Microbiol. Biotechnol. 2012, 96, 1243–1252. [CrossRef] 24. Sheng, B.; Xu, J.; Ge, Y.; Zhang, S.; Wang, D.; Gao, C.; Ma, C.; Xu, P. Enzymatic resolution by a D-lactate oxidase catalyzed reaction for (S)-2-hydroxycarboxylic acids. ChemCatChem 2016, 8, 2630–2633. [CrossRef] 25. Montella, C.; Bellsolell, L.; Pérez-Luque, R.; Badía, J.; Baldoma, L.; Coll, M.; Aguilar, J. Crystal structure of an iron-dependent group III dehydrogenase that interconverts L-lactaldehyde and L-1,2-propanediol in Escherichia coli. J. Bacteriol. 2005, 187, 4957–4966. [CrossRef] 26. Iding, H.; Siegert, P.; Mesch, K.; Pohl, M. Application of α-keto acid decarboxylases in biotransformations. Biochim. Biophys. Acta Protein Struct. Mol. Enzym. 1998, 1385, 307–322. [CrossRef] 27. Wang, X.; Miller, E.N.; Yomano, L.P.; Zhang, X.; Shanmugam, K.T.; Ingram, L.O. Increased furfural tolerance due to overexpression of NADH-dependent oxidoreductase FucO in Escherichia coli strains engineered for the production of ethanol and lactate. Appl. Environ. Microbiol. 2011, 77, 5132–5140. [CrossRef] 28. Zhu, Y.; Lin, E.C. L-1,2-propanediol exits more rapidly than L-lactaldehyde from Escherichia coli. J. Bacteriol. 1989, 171, 862–867. [CrossRef] 29. Wang, Y.; Xian, M.; Feng, X.; Liu, M.; Zhao, G. Biosynthesis of ethylene glycol from D-xylose in recombinant Escherichia coli. Bioengineered 2018, 9, 233–241. [CrossRef][PubMed] 30. Feng, J.; Zhang, Y.; Xiong, W.; Ding, H.; He, B. Hydrogenolysis of glycerol to 1,2-propanediol and ethylene glycol over Ru- Co/ZrO2 catalysts. Catalysts 2016, 6, 51. [CrossRef] 31. Pereira, B.; Zhang, H.; De Mey, M.; Lim, C.G.; Li, Z.; Stephanopoulos, G. Engineering a novel biosynthetic pathway in Escherichia coli for production of renewable ethylene glycol. Biotechnol. Bioeng. 2016, 113, 376–383. [CrossRef] 32. Zhang, Z.; Yang, Y.; Wang, Y.; Gu, J.; Lu, X.; Liao, X.; Shi, J.; Kim, C.H.; Lye, G.; Baganz, F.; et al. Ethylene glycol and glycolic acid production from xylonic acid by Enterobacter cloacae. Microb. Cell Fact. 2020, 19, 1–16. [CrossRef] 33. Pereira, B.; Li, Z.-J.; De Mey, M.; Lim, C.G.; Zhang, H.; Hoeltgen, C.; Stephanopoulos, G. Efficient utilization of pentoses for bioproduction of the renewable two-carbon compounds ethylene glycol and glycolate. Metab. Eng. 2016, 34, 80–87. [CrossRef] 34. Chae, T.U.; Choi, S.Y.; Ryu, J.Y.; Lee, S.Y. Production of ethylene glycol from xylose by metabolically engineered Escherichia coli. AICHE J. 2018, 64, 4193–4200. [CrossRef] 35. Cabulong, R.B.; Chung, W.-J.; Ramos, K.R.M.; Nisola, G.M.; Lee, W.-K.; Lee, C.R.; Chung, W.-J. Enhanced yield of ethylene glycol production from d-xylose by pathway optimization in Escherichia coli. Enzym. Microb. Technol. 2017, 97, 11–20. [CrossRef] 36. You, C.; Shi, T.; Li, Y.; Han, P.; Zhou, X.; Zhang, Y.-H.P. An in vitro synthetic biology platform for the industrial biomanufacturing of myo-inositol from starch. Biotechnol. Bioeng. 2017, 114, 1855–1864. [CrossRef] 37. Rollin, J.A.; Del Martin del Campo, J.; Myung, S.; Sun, F.; You, C.; Bakovic, A.; Castro, R.; Chandrayan, S.K.; Wu, C.-H.; Adams, M.W.; et al. High-yield hydrogen production from biomass by in vitro metabolic engineering: Mixed sugars coutilization and kinetic modeling. Proc. Natl. Acad. Sci. USA 2015, 112, 4964–4969. [CrossRef][PubMed]