Biocatalytic Conversion of Short-Chain Fatty Acids to Corresponding Alcohols in Escherichia Coli

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Article Biocatalytic Conversion of Short-Chain Fatty Acids to Corresponding Alcohols in Escherichia coli

Li-Jen Lin 1 , Mukesh Saini 2, Chung-Jen Chiang 3,* and Yun-Peng Chao 2,4,5,*

1 School of Chinese Medicine, College of Chinese Medicine, China Medical University, No. 91, Hsueh-Shih Road, Taichung 40402, Taiwan; [email protected] 2 Department of Chemical Engineering, Feng Chia University, 100 Wenhwa Road, Taichung 40724, Taiwan; [email protected] 3 Department of Medical Laboratory Science and Biotechnology, China Medical University, No. 91, Hsueh-Shih Road, Taichung 40402, Taiwan 4 Department of Health and Nutrition Biotechnology, Asia University, Taichung 41354, Taiwan 5 Department of Medical Research, China Medical University Hospital, Taichung 40447, Taiwan * Correspondence: [email protected] (C.-J.C.); [email protected] (Y.-P.C.); Tel.: +886-4-22-053-366 (ext. 7227) (C.-J.C.); +886-4-24-517-250 (ext. 3677) (Y.-P.C.); Fax: +886-4-22-057-414 (C.-J.C.); +886-4-24-510-890 (Y.-P.C.)

Abstract: Advanced biofuels possess superior characteristics to serve for gasoline substitutes. In this study, a whole cell biocatalysis system was employed for production of short-chain alcohols from corresponding fatty acids. To do so, Escherichia coli strain was equipped with a biocatalytic pathway consisting of endogenous atoDA and Clostridium acetobutylicum adhE2. The strain was further reprogrammed to improve its biocatalytic activity by direction the glycolytic ﬂux to acetyl-

CoA and recycling acetate. The production of 1-propanol and n-pentanol were exempliﬁed with the engineered strain. By substrate (glucose and propionate) feeding, the strain enabled production

Citation: Lin, L.-J.; Saini, M.; Chiang, of 5.4 g/L 1-propanol with productivity reaching 0.15 g/L/h. In addition, the strain with a heavy C.-J.; Chao, Y.-P. Biocatalytic inoculum was implemented for the n-pentanol production from n-pentanoic acid. The production Conversion of Short-Chain Fatty titer and productivity ﬁnally attained 4.3 g/L and 0.86 g/L/h, respectively. Overall, the result Acids to Corresponding Alcohols in indicates that this developed system is useful and effective for biocatalytic production of short- Escherichia coli. Processes 2021, 9, 973. chain alcohols. https://doi.org/10.3390/pr9060973 Keywords: short-chain fatty alcohols; short-chain fatty acids; biocatalysis Academic Editor: Juan Francisco García Martín

Received: 13 May 2021 1. Introduction Accepted: 29 May 2021 Published: 31 May 2021 Our daily life which mainly depends on fossil resources has caused the enormous emission of greenhouse gases, consequently leading to global climate change. It urges us to

Publisher’s Note: MDPI stays neutral search for sustainable energy alternatives and environment-friendly chemicals [1]. Biofuels with regard to jurisdictional claims in that are produced from renewable feedstock apparently fulﬁll the need. Ethanol appears to published maps and institutional afﬁl- be a representative biofuel. However, it has unfavorable properties including low energy iations. density, high vapor pressure, and hygroscopicity [2]. Advanced biofuels involving longer chain alcohols (C3–C6) are of particular interest because they have superior characteristics applied for gasoline substitutes. For instance, n-butanol (C4) which is blended with gasoline at any concentrations readily serves as the transportation fuel. There is no need to modify the existing pipeline infrastructure for transport of n-butanol [3]. The fuel property of Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland. isobutanol is similar to n-butanol whereas it possesses higher octane number. Isopentanol This article is an open access article (C5) is a branched-chain alcohol and shares similarity in the physiochemical property with distributed under the terms and gasoline [4]. In particular, it has a higher volumetric energy density than n-butanol. As a conditions of the Creative Commons linear-chain alcohol, n-pentanol (C5) is exploited as an additive in diesel [5]. It results in Attribution (CC BY) license (https:// diesel fuel with a better performance in terms of fuel combustion and particulate emission. creativecommons.org/licenses/by/ Likewise, hexanol (C6) in the form of a gasoline blend improves the fuel property [6]. In 4.0/). addition to a gasoline additive, 1-propanol (C3) serves as a precursor for the synthesis of

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propylene. Propylene is used for the production of plastics, and it is an important platform chemical in industry as well [7]. Bulk chemicals derived from propylene include propylene oxide, acrylonitrile, cumene, butyraldehyde, and acrylic acid. Naturally-occurring microbes harbor inherent metabolic pathways for the synthesis of advanced alcohols. The best known example is the production of n-butanol in Clostridium species which undergo the mixed acetone-butanol-ethanol (ABE) fermentation [8]. The ABE fermentation proceeds with the acidogenesis phase, followed by the solventogenesis phase [9]. However, the sluggish growth and sporulation of Clostridium usually render the operation difficult and complicated. Propionibacteria natively produce propionic acid through the Wood-Werkman pathway [10]. The production of 1-propanol occurs when the carbon source changes to the more reduced one for fermentation. Nevertheless, propionic acid still remains the major product. In the absence of oxygen, Clostridium propionicum enables utilization of ethanol or lactate via the acrylate pathway [11]. The fermentation product ends up with propionic acid and acetic acid while 1-propanol is marginally produced. In addition, C. carboxidivorans is an acetogen that relies on the Wood-Ljungdahl pathway to grow on syngas (CO or CO2 and H2)[12]. By lowering the metabolic activity, the acetogenic fermentation resulted in the improvement in the production of ethanol, butanol, and hexanol. However, this production process is afflicted by low titer and productivity. The recent advance in the technology of synthetic biology has successfully transformed various microbes into producers capable of synthesizing advanced biofuels. Recognized as the biotechnology workhorse, Escherichia coli has been subject to rational reprogramming of metabolic pathways, mainly including the 2-ketoacid pathway, the fatty acid synthesis pathway, and the reverse β-oxidation pathway [13,14]. The engineered E. coli generally shows promise for fermentative production of C3–C6 alcohols. Nevertheless, the technical bottleneck still exists and remains to be overcome. To address this issue, we have recently designed a synthetic consortium for production of n-butanol [15]. In this system, an E. coli strain responsible for conversion of butyrate to n-butanol was equipped with a biocatalytic route consisting of endogenous atoDA and C. acetobutylicum adhE2. With supplemented butyrate, this butyrate-conversion strain effectively produced 6.2 g/L n- butanol. In this study, the biocatalysis biocatalysis system based on this strain was exploited for the synthesis of other C3–C6 alcohols from corresponding fatty acids. The central metabolism of the strain was further refined to improve the biocatalytic activity. The result shows that this platform system is useful and effective for production of short-chain fatty alcohols.

2. Materials and Methods 2.1. Strain Construction The strain development started with BuT-3EA strain. This strain was derived from BuT-3E strain and carried the enhanced level of acs [15]. The strain’s modiﬁcation was carried out as follows. Following the pervious study [16], the DNA cassette was ampli- ﬁed from pPR-aceE plasmid with RC12060/RC12086 primers. The PCR DNA was then introduced into the strain by electroporation. By the λRed-mediated homologous recombi- nation, the DNA cassette was integrated into the strain’s genome. The inserted LE*-kan-RE* cassette was removed by the act of Cre. This construction resulted in the strain carrying the aceEF operon under the control of the λPL promoter (PλPL). Moreover, pLam-LpdA* plasmid which contained the NADH-insensitive lpdA mutant (lpdA*) fused to PλPL (PλPL- lpdA*) was used for the enhanced expression of lpdA. The DNA containing PλPL-lpdA* was integrated into the λ attachment site of the strain, followed by removal of the inserted marker. The resulting strain was renamed BuT-3EP.

2.2. Fatty Alcohol Production E. coli strains were grown on Luria-Bertani medium at 37 ◦C overnight. The bacterial growth was measured with a spectrometer set at 550 nm (OD550). The overnight culture was seeded to Erlenmeyer ﬂasks (125 mL) containing M9Y medium (20 mL) supplemented Processes 2021, 9, 973 3 of 11

with glucose (10 g/L) and fatty acids (4 g/L). The medium was composed of Na2HPO4 (6 g/L), KH2PO4 (3 g/L), NaCl (0.5 g/L), NH4Cl (1 g/L), MgSO4 (1 mM), CaCl2 (0.1 mM), and yeast extract (5 g/L). The initial cell density was maintained at OD550 of 0.2. The shake- ﬂask culture was conducted under the oxygen-limited condition according to the previous study [15]. The biocatalytic production of fatty alcohols was terminated at 48 h. The mode of substrate feeding was performed by adding extra glucose (10 g/L) and propionic acid (4 g/L) to the culture medium at 24 h. Incubated with n-pentanoic acid (6 g/L), the strain with the initial cell density at OD550 of 9 was applied for the n-pentanol production.

2.3. Analytic Methods The analysis of glucose, alcohols, and organic acids essentially followed the reported protocol [15]. Glucose was determined using high-performance liquid chromatography (HPLC) with the ICSep ICE-ION-300 column (Transgenomic, Omaha, NE, USA). HPLC with the reflective index (RID-10A, Shimadzu, Japan) was used for the measurement of organic acids. Alcohols were analyzed by gas chromatograph (Trace 1300, Thermo Scientific, Waltham, MA, USA) which was equipped with flam ionization detector and a DB-WAX capillary column. The oven temperature was initially held at 50 ◦C for 1 min and raised to 150 ◦C with a gradient of 15 ◦C/min, and 150 ◦C was maintained for 5 min. The injector and detector were maintained at 250 ◦C

3. Results 3.1. Production of Fatty Alcohols In this study, a whole cell biocatalysis system was proposed to produce short-chain fatty alcohols. BuT-3E strain enabled production of n-butanol from n-butyric acid and was implemented for the engineering purpose [15]. As indicated in Figure1, this strain was equipped with a synthetic pathway consisting of endogenous atoDA and Clostridium adhE2. Acetoacetyl-CoA transferase encoded by atoDA has a physiological function for activation of short-chain fatty acids (C4–C6) to corresponding thioesters associated with acetic acid [17]. The subsequent conversion of thioesters to respective alcohols proceeds through the reaction catalyzed by alcohol dehydrogenase (encoded by adhE2). In addition, the undesired pathways were blocked to conserve NADH and to reduce the production of waste products. It was intriguing to learn the performance of BuT-3E strain for production of alter- native fatty alcohols. Therefore, the shake-ﬂask culture was conducted with the medium containing short-chain fatty acids (C3–C7) under the oxygen-limited condition. At the end of the fermentation, the strain produced 1-propanol, isobutanol, n-pentanol, isopentanol, and n-hexanol with a titer reaching 2.3, 1.8, 1.5, 1.2 and 1.3 g/L, respectively (Figure2a). As shown in Figure2b, propionic acid (3.4 g/L), isobutanoic acid (2.4 g/L), n-pentanoic acid (2 g/L), isopentanoic acid (1.8 g/L), and n-hexanoic acid (1.7 g/L) were consumed in the strain to serve as the precursor for the synthesis of alcohols. Consequently, the conversion yield of 1-propanol, isobutanol, n-pentanol, isopentanol, and n-hexanol based on corresponding acids accounts for 83.4%, 89.2%, 86.9%, 77.3%, and 87.0% of the theoretical yield (ca., 0.81, 0.84, 0.86, 0.88 g/g), respectively. The result indicates that BuT-3E strain shows promise for production of C3–C6 alcohols. In addition, n-heptanol was not detected. This is attributed to the failure of atoDA in activation of heptanoic acid (Figure2b). Processes 2021, 9, x FOR PEER REVIEW 4 of 11

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Figure 1. The biocatalytic pathway leading to fatty alcohols in E. coli. The biocatalytic pathway leading to fatty alcohols Figure 1. The frombiocatalytic short-chain pathway fatty acids waslead introduceding to fatty into E.alcohols coli by metabolic in E. coli engineering.. The biocatalytic Undesired pathways pathway were leading blocked to by fatty alcohols from short-chaindeletion fatty of responsibleacids was genes introduced as marked into by “X”. E. coli The genesby metabolic involved in engineering. the metabolic pathway: UndesiredaceEF -pathwayslpdA*: pyruvate were blocked by deletion of responsibledehydrogenase genes complex; as acsmarked, acetyl-CoA by synthetase;“X”. TheadhE genes, aldehyde-alcohol involved in dehydrogenase; the metabolicadhE2 ,pathway: butyraldehyde-butanol aceEF-lpdA*: pyruvate dehydrogenasedehydrogenase; complex;atoDA acs,, acetoacetyl-CoAacetyl-CoA transferase;synthetase;ldhA , lactateadhE dehydrogenase;, aldehyde-alcoholfrdA, subunit dehydrogenase; of fumarate reductase; adhE2gltA, , butyralde- citrate synthase; poxB, pyruvate oxidase; pta, phosphate acetyltransferase. Abbreviations: ACE, acetate; CIT, citrate; EtOH, hyde-butanol dehydrogenase; atoDA, acetoacetyl-CoA transferase; ldhA, lactate dehydrogenase; frdA, subunit of fumarate ethanol; Glc, glucose; LAC, lactate; OAA, oxaloacetate; PEP, phosphoenolpyruvate; PYR, pyruvate; SUC, succinate; 2-MC, reductase; gltA2-methylcitrate., citrate synthase; poxB, pyruvate oxidase; pta, phosphate acetyltransferase. Abbreviations: ACE, acetate; CIT, citrate; EtOH, ethanol; Glc, glucose; LAC, lactate; OAA, oxaloacetate; PEP, phosphoenolpyruvate; PYR, pyruvate; SUC, succinate; 2-MC, 2-methylcitrate.

It was intriguing to learn the performance of BuT-3E strain for production of alter- native fatty alcohols. Therefore, the shake-flask culture was conducted with the medium containing short-chain fatty acids (C3–C7) under the oxygen-limited condition. At the end of the fermentation, the strain produced 1-propanol, isobutanol, n-pentanol, isopentanol, and n-hexanol with a titer reaching 2.3, 1.8, 1.5, 1.2 and 1.3 g/L, respectively (Figure 2a). As shown in Figure 2b, propionic acid (3.4 g/L), isobutanoic acid (2.4 g/L), n-pentanoic acid (2 g/L), isopentanoic acid (1.8 g/L), and n-hexanoic acid (1.7 g/L) were consumed in the strain to serve as the precursor for the synthesis of alcohols. Conse- quently, the conversion yield of 1-propanol, isobutanol, n-pentanol, isopentanol, and n-hexanol based on corresponding acids accounts for 83.4%, 89.2%, 86.9%, 77.3%, and 87.0% of the theoretical yield (ca., 0.81, 0.84, 0.86, 0.88 g/g), respectively. The result indicates that BuT-3E strain shows promise for production of C3–C6 alcohols. In addition, n-heptanol was not detected. This is attributed to the failure of atoDA in activation of heptanoic acid (Figure 2b).

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Alcohols Acetate OD 8

2.5 7

6 2.0 5 1.5 4 550 OD 1.0 3

2 0.5

Alcohols and acetate (g/L) 1

0.0 0 Propanol Isobutanol Pentanol Isopentanol Hexanol Heptanol

(a) Glucose Acids 10

2 Consumed glucose Consumed and fatty acids (g/L) 0 Propanoic Isobutanoic Pentanoic Isopentanoic Hexanoic Heptanoic acid acid acid acid acid acid

(b)

FigureFigure 2. 2.Production Production of of short-chain short-chain alcohols alcohols in in engineered engineeredE. E. coli coli.. The The fermentation fermentation was was conducted conducted with BuT-3E strain in the presence of glucose (10 g/L) and short-chain fatty acids (4 g/L) as indi- with BuT-3E strain in the presence of glucose (10 g/L) and short-chain fatty acids (4 g/L) as indicated cated for 24 h. The experiments were conducted in triplicate. The fermentation products and the for 24 h. The experiments were conducted in triplicate. The fermentation products and the cell cell density (OD550) were determined at the end of the fermentation (a). The consumption of glucose densityand fatty (OD acids550) werewere determinedmeasured for at thefurther end analyses of the fermentation (b). (a). The consumption of glucose and fatty acids were measured for further analyses (b).

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3.2. Propanol Production by Substrate Feeding As illustrated above, BuT-3E strain enabled production of C3-C6 fatty alcohols with Processes 2021, 9, 973 a high conversion yield. Nevertheless, the alcohol production decreased with an6 ofincrease 11 in the chain length. Taking 1-propanol as an example, our next task was undertaken to improve the production yield. It is well recognized that short-chain fatty acids with high solubility 3.2.are Propanol detrimental Production to bymicrobes Substrate Feedingbecause they penetrate into cell membrane at a high concentration,As illustrated which above, in turn BuT-3E damages strain enabledcell membrane production [18]. of C3-C6 The fatty bacterial alcohols growth with a was greatly inhibitedhigh conversion by propionic yield. Nevertheless, acid at a level the alcohol higher production than 4 g/L decreased (data not with shown). an increase There- in the chain length. Taking 1-propanol as an example, our next task was undertaken to fore, the fermentationimprove the production of 1-propanol yield. It was is well cond recognizeducted with that short-chain the substrate fatty acidsfeeding. with highIn a similar way, BuT-3Esolubility strain are detrimental was first tocultivated microbes becausewith medium they penetrate containing into cell 10 membraneg/L glucose at aand 4 g/L propionichigh concentration,acid. At 24 h, which 10 g/L in turnglucose damages and cell 4 g/L membrane propionic [18]. acid The bacterial were additionally growth was fed to the culture.greatly As inhibited a result, by propionicthe 1-propanol acid at a levelproduction higher than leveled 4 g/L (data off notat 36 shown). h and Therefore, reached 4.2 the fermentation of 1-propanol was conducted with the substrate feeding. In a similar g/L (Figureway, 3a). BuT-3E The strainstrain was consumed ﬁrst cultivated around with medium13 g/L glucose containing and 10 g/L 6 g/L glucose propionic and 4 g/L acid at the end (Figurepropionic 3b). acid. It Atleads 24 h, 10to g/Lthe glucoseconversion and 4 g/Lyield propionic based acid on werepropionic additionally acid fed reaching to 86.4% of thethe culture.theoretical As a result,yield. the In 1-propanoladdition, production the cell density leveled off was at 36 not h and affected reached by 4.2 the g/L extra addition of(Figure glucose3a). The(Figure strain 3b). consumed The result around indicates 13 g/L glucose that and the 6 cell g/L propionicgrowth is acid limited at the and, however, endthe (Figurestrain3 remainsb). It leads metabolically to the conversion acti yieldve basedto provide on propionic acetyl-CoA acid reaching for the 86.4% synthesis of the theoretical yield. In addition, the cell density was not affected by the extra addition of of 1-propanol.glucose (Figure3b). The result indicates that the cell growth is limited and, however, the strain remains metabolically active to provide acetyl-CoA for the synthesis of 1-propanol.

(a)

Figure 3. Cont.

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(b)

Figure 3. ProductionFigure 3. Production of 1-propanol of 1-propanol by the by approach the approach of of substrate substratefeeding. feeding. The The fermentation fermentation of BuT-3E of BuT-3E strainstrain was was carried carried out with with glucose glucose (10 g/L)(10 g/L) and propionate and propionate (4 g/L). At (424 g/L). h, the At substrate 24 h, the containing substrate containing glucose (10 (10 g/L) g/L) and and propionate propionate (4 g/L) (4 g/L) was addedwas added to the culture to the mediumculture andmedium the fermentation and the fermentation resultresult waswas analyzed analyzed at aat predetermined a predetermi interval.ned interval. The experiment The experiment was conducted was in conducted triplicate. The in fermentation products and the cell density (OD550) were determined at the end of the fermentation triplicate. The fermentation products and the cell density (OD550) were determined at the end of the (a). The consumption of glucose and fatty acids were measured for further analyses (b). fermentation (a). The consumption of glucose and fatty acids were measured for further analyses (b). 3.3. Improvement of Propanol Production Acetic acid appears to be an associated product in the synthetic pathway of fatty 3.3. Improvementalcohols of in Propanol the strain. Production However, it disables the growth of E. coli at a low level and imparts an inhibitory effect on methionine biosynthesis [19]. Acetyl-CoA synthetase (encoded by Aceticacs acid) activates appears acetate to to be acetyl-CoA an associated at the expense product of ATP in andthe playssynthetic a physiological pathway role of in fatty alcohols inscavenging the strain. acetate However, under the it anaerobicdisables conditionthe growth [20]. Itof also E. coli involves at a inlow the level 2-methylcitrate and imparts an inhibitory(2-MC) effect cycle on for methionine oxidation of propionate biosynthes andis produce [19]. Acetyl-CoA propionyl-CoA synthetase from propionate (encoded [21]. by acs) activatesIn addition, acetate pyruvateto acetyl-CoA oxidase (encodedat the expense by poxB )of catalyzes ATP and oxidative plays decarboxylation a physiological of role pyruvate to acetate. Therefore, the expression of acs and aceEF operon were enhanced to in scavengingimprove acetate the fermentation under the performance anaerobic of the condition strain. The [20]. increased It also activity involves of pyruvate in the 2-methylcitratedehydrogenase (2-MC) (encoded cycle for by aceEFoxidation) directs of pyruvate propionate to acetyl-CoA and produce and away frompropionyl-CoA the poxB from propionatepathway. [21]. The recruitmentIn addition, of ACSpyruvate facilitates oxidase re-utilization (encoded of acetate. by poxB The) resulting catalyzes strain oxidative decarboxylationwas designated of as pyruvate BuT-3EP. The to substrate-feedingacetate. Therefore, mode wasthe thenexpression performed of with acs BuT-3EP and aceEF operon werestrain. enhanced As a consequence, to improve the strainthe fermen consumedtation propionic performance acid of 7.4 of g/L the and strain. produced The in- 1-propanol of 5.4 g/L at 36 h (Figure4a,b). The conversion yield based on propionic acid creased activityaccounts of for pyruvate 90% of the dehydrogenase theoretical yield. The(encoded production by ofaceEF acetic) aciddirects reduced pyruvate to 2.4 g/L to acetyl-CoA andfrom 4away g/L (i.e., from BuT-3E the strain poxB in Figure pathway.3a) at the The end. Nevertheless,recruitment the of bacterial ACS growth facilitates re-utilizationwas arrestedof acetate. and not The further resulting improved strain by the supplementwas designated of extra glucose.as BuT-3EP. It is likely The that substrate-feedingthe toxicity mode of was acetic then acid in performed part limits the with strain’s BuT-3EP growth. Moreover, strain. As the combineda consequence, function the of AtoDA and ACS is expected to raise the propionyl-CoA level, which likely perturbs the strain consumedCoA homeostasis propionic and acid increases of 7.4 the g/L production and produced of 2-MC via 1-propanol the 2-MC pathway. of 5.4 2-MCg/L at is 36 h (Figure 4a,b).known The to beconversion a potent inhibitor yield ofbased citrate on synthase propionic in the tricarboxylicacid accounts acid (TCA)for 90% cycle of [22 the]. theoretical yield. The production of acetic acid reduced to 2.4 g/L from 4 g/L (i.e., BuT-3E strain in Figure 3a) at the end. Nevertheless, the bacterial growth was arrested and not further improved by the supplement of extra glucose. It is likely that the toxicity of acetic acid in part limits the strain’s growth. Moreover, the combined function of AtoDA and ACS is expected to raise the propionyl-CoA level, which likely perturbs the CoA homeostasis and increases the production of 2-MC via the 2-MC pathway. 2-MC is known to be a potent inhibitor of citrate synthase in the tricarboxylic acid (TCA) cycle [22].

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Propanol Acetate OD 6 8

5 6 4

3 4 550 OD 2 2 1 Propanol andacetate (g/L)

0 0 24 36 48 Time (h)

(a) Glucose Propionate 14

Consumed glucose and propionate (g/L) 2

0 24 36 48 Time (h)

(b)

FigureFigure 4. 4.ProductionProduction of 1-propanol of 1-propanol by the by genetically-modified the genetically-modiﬁed strain. strain.Derived Derived from BuT-3E from strain, BuT-3E strain, BuT-3EP strain was employed for the fermentation in the presence of glucose (10 g/L) and propi- BuT-3EP strain was employed for the fermentation in the presence of glucose (10 g/L) and propionate onate (4 g/L). At 24 h, the substrate containing glucose (10 g/L) and propionate (4 g/L) was added to (4 g/L). At 24 h, the substrate containing glucose (10 g/L) and propionate (4 g/L) was added to the culture medium and the fermentation result was analyzed at a predetermined interval. The experiment was conducted in triplicate. The fermentation products and the cell density (OD ) were 550 determined at the end of the fermentation (a). The consumption of glucose and fatty acids were measured for further analyses (b). Processes 2021, 9, x FOR PEER REVIEW 9 of 11

the culture medium and the fermentation result was analyzed at a predetermined interval. The Processes 2021, 9, 973 experiment was conducted in triplicate. The fermentation products and the cell density (OD550) 9 of 11 were determined at the end of the fermentation (a). The consumption of glucose and fatty acids were measured for further analyses (b).

3.4.3.4. n-Pentanol n-Pentanol Production Production by byHeavy Heavy Inoculum Inoculum OurOur last last task task was was undertaken undertaken to improve to improve productivity. productivity. This Thisissue issue was addressed was addressed by implementationby implementation of the of fermentation the fermentation mode mode based based on the on heavy the heavy inoculum. inoculum. In this In case, this the case, n-pentanolthe n-pentanol production production was chosen was chosen for illust forration. illustration. The fermentation The fermentation was carried was carried out us- out ingusing BuT-3EP BuT-3EP strain strain with with the thecell celldensity density reaching reaching 9 at 9 OD at OD550 while550 while 10 g/L 10 g/Lglucose glucose andand 6 g/L6 g/Ln-pentanoic n-pentanoic acid were acid weresupplemented supplemented in the inmedium. the medium. As shown As shownin Figure in 5, Figure the strain5, the producedstrain produced 4.3 g/L n-pentanol 4.3 g/L n-pentanol at the expense at the of expense 5.5 g/L of n-pentanoic 5.5 g/L n-pentanoic acid at 5 h. acid Conse- at 5 h. quently,Consequently, the conversion the conversion yield based yield on based n-pent onanoic n-pentanoic acid accounts acid accounts for 90.5% for of 90.5% the theo- of the reticaltheoretical yield. yield.

OD Pentanoate Glucose Pentanol 16 16 10 14 14 8 12 12 10 6 10

550 8 8 4 6 OD 6 4 2 (g/L) Pentanol 4 2

Pentanoate/Glucose (g/L) Pentanoate/Glucose 0 2 0 0 0123456 Time (h)

FigureFigure 5. 5.ImprovedImproved production production of ofn-pentanol. n-pentanol. The The ferm fermentationentation of BuT-3EP of BuT-3EP strain strain was was carried carried out out withwith glucose glucose (10 (10 g/L) g/L) and and n-pentanote n-pentanote (6 g/L). (6 g/L). The The fermentation fermentation was was then then followed followed along along the thetime time course.course. The The experiment experiment was was conducted conducted in intriplicate. triplicate.

4. 4.Discussion Discussion MetabolicMetabolic engineering engineering is isan an enabling enabling techno technologylogy that that efficiently efficiently reprograms reprograms living living cellscells for for the the desired desired trait. trait. This This technology technology has has been been applied applied for for the the microbial microbial fermentation fermentation productionproduction of ofshort-chain short-chain alcohols. alcohols. E. coliE. colistillstill remains remains the top the topchoice choice for engineering for engineering to developto develop a production a production scheme scheme of industrial of industrial usefulness. usefulness. As Asexemp exemplifiedlified by by 1-propanol 1-propanol production,production, two two metabolic metabolic pathways pathways leadingleading toto the the formation formation of of 2-ketobutyrate 2-ketobutyrate (2-KB) (2-KB) were wereconstructed constructed in E. in coli E.. Thecoli. synthesisThe synthesis pathway pathway of L-threonine of L-threonine involving involvingthrABC thrABCand ilvA andwas ilvAmanipulated was manipulated to direct to the direct glycolytic the glycolytic flux from flux oxaloacetate from oxaloacetate (OAA) to (OAA) 2-KB. The to 2-KB. recruitment The recruitmentof cimA and ofleuBCD cimA andconstituted leuBCD theconstituted heteroglogous the heteroglogous citramalate pathway citramalate responsible pathway for re- the sponsiblesynthesis for of the 2-KB synthesis from pyruvate of 2-KB (PYR). from Moreover,pyruvate (PYR). the inactivation Moreover, of the four inactivation engaged genes of fourconserved engaged the genes intracellular conserved 2-KB the intracellular pool which was 2-KB further pool which converted was tofurther 1-propanol converted through to the synthesis pathway consisting of kivd and adhE. As a result, the engineered E. coli utilized 1-propanol through the synthesis pathway consisting of kivd and adhE. As a result, the glucose to produce 1-propanol of 8 g/L with productivity reaching 0.12 g/L/h [23]. In engineered E. coli utilized glucose to produce 1-propanol of 8 g/L with productivity another study, the ‘sleeping beauty mutase’ operon was activated by overexpression of sbm, reaching 0.12 g/L/h [23]. In another study, the ‘sleeping beauty mutase’ operon was ac- ygfD, and ygfG. Equipped with the additional function of sucCD and adh2, E. coli strain was tivated by overexpression of sbm, ygfD, and ygfG. Equipped with the additional function subject to fed-batch fermentation and enabled production of 7 g/L 1-propanol based on of sucCD and adh2, E. coli strain was subject to fed-batch fermentation and enabled pro- glycerol with productivity of 0.04 g/L/h [24]. To synthesize 1-propanol, the methylglyoxal duction of 7 g/L 1-propanol based on glycerol with productivity of 0.04 g/L/h [24]. To bypass pathway was modified by expression of heterologous budC and ppdABC. This modified pathway leads to the formation of 1,2-propanediol (1,2-PDO) as an intermediate metabolite. The resulting strain produced 0.25 g/L 1-propanol from glucose and the

productivity was 0.005 g/L/h [25]. The 1-propanol production was further optimized by using the co-cultivation strategy of two E. coli strains responsible for production of Processes 2021, 9, 973 10 of 11

and conversion of 1,2-PDO, respectively. Finally, the result gave 1-propanol of 3 g/L and productivity of 0.014 g/L/h [26]. In contrast to these studies, this work proposed a whole cell biocatalysis system. The core of this system relies on the native atoDAEB operon which is engaged in the degradation of C4-C6 fatty acids [27]. The physiological function of AtoDA activates acetoacetate to acetoacetyl-CoA. Fed with propionic acid, BuT-3E strain bearing functional AtoDA enabled production of 1-propanol (Figure2a,b). The result indicates that AtoDA functions to activate propionate to propionly-CoA which is subsequently oxidized to 1-propanol by AdhE2. The producer strain was improved by the approach of metabolic engineering, which aims to recycle acetic acid and reﬁne the glycolysis pathway for availability of acetyl-CoA (Figure1). By the substrate-feeding strategy, the engineered strain (i.e., BuT-3EP) produced 5.4 g/L 1-propanol and attained productivity of 0.15 g/L/h (Figure4a,b). It is apparent that our proposed system shows high efﬁciency in production of 1-propanol. The application of this biocatalysis system was further illustrated with the production of n-pentanol. Initiated with a heavy inoculum, the batch culturing of BuT-3EP strain produced 4.3 g/L n-pentanol with productivity reaching 0.86 g/L/h (Figure5). Intensive efforts have been devoted to the fermentative production of n-pentanol. In combination with the 2-ketoacid pathway, the reversed β-oxidation pathway in the strain was employed for the synthesis of n-pentanol with a titer and productivity reaching 0.4 g/L and 0.004 g/L/h, respectively [28]. However, the production of n-pentanol was associated with many alcohol byproducts of various chain length. Another study reported the implementation of the iterative elongation cycle involved in the 2-ketoacid pathway. To improve the n-pentanol synthesis, Lactococcus lactis ketoisovalerate decarboxylase (Kivd) was subject to mutation. As a result, the Kivd mutant strain enabled production of pentanol accounting for 90% of the total alcohol content. The application of in situ extraction with oleyl alcohol led to the production titer of 4.3 g/L and productivity of 0.029 g/L/h [29]. Nevertheless, this previous work generally has the disadvantage of low productivity and product promiscuity. In summary, this study proposed a promising system for biocatalytic production of short-chain fatty alcohols. The developed strain is, however, susceptible to toxicity at a high concentration of fatty acids and fatty alcohols. The method of metabolic evolution is useful for selection of mutant strains that tolerate high levels of fatty acids and alcohols [30]. Meanwhile, the approach by in situ extraction of alcohols facilitates the bacterial survival. The usefulness of this bioconversion platform would be greatly acknowledged by the successful implementation of these combined strategies to reshape the producer strain with the desired trait.

Author Contributions: Writing—original draft preparation, L.-J.L.; methodology and analysis, M.S.; supervision, C.-J.C.; writing—review and editing, Y.-P.C. All authors have read and agreed to the published version of the manuscript. Funding: This work is supported by Ministry of Science and Technology (MOST 109-2320-B-036-MY3 and 108-2221-E-035-052-MY3), Taiwan. Conﬂicts of Interest: The authors declare no conﬂict of interest.

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