High-level production of 4-hydroxyvalerate from levulinic acid

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High-level production of 4-hydroxyvalerate from levulinic acid via whole-cell biotransformation decoupled from cell metabolism Doyun Kim, Chandran Sathesh-Prabu, Young JooYeon, and Sung Kuk Lee J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.9b04304 • Publication Date (Web): 02 Sep 2019 Downloaded from pubs.acs.org on September 2, 2019

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Journal of Agricultural and Food Chemistry

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High-level production of 4-hydroxyvalerate from levulinic acid via whole-cell

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biotransformation decoupled from cell metabolism

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Doyun Kim a, Chandran Sathesh-Prabu b, Young JooYeon c, and Sung Kuk Lee a, b,*

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a

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Technology (UNIST), Ulsan 44919, Republic of Korea

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b

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Technology (UNIST), Ulsan 44919, Republic of Korea

Department of Biomedical Engineering, Ulsan National Institute of Science and

Department of Chemical Engineering, Ulsan National Institute of Science and

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c

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Gangneung 25457, Republic of Korea

Department of Biochemical Engineering, Gangneung-Wonju National University,

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*Corresponding author. Address: Department of Chemical Engineering, Ulsan National

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Institute of Science and Technology (UNIST), Ulsan 44919, Republic of Korea.

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Tel.: +82 52 217 2514; Fax: +82 52 217 3009

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E-mail address: [email protected] (S.K.Lee)

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ACS Paragon Plus Environment


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ABSTRACT Gamma-hydroxyvalerate (4HV) is an important monomer used to produce

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various valuable polymers and products. In this study, an engineered 3-hydroxybutyrate

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dehydrogenase (3HBDH*) that can convert levulinic acid (LA) into 4HV was co-

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expressed with a cofactor (NADH) regeneration system mediated by an NAD+-

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dependent formate dehydrogenase (CbFDH) in an Escherichia coli strain MG1655. The

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resulting strain produced 23-fold more 4HV in a shake flask. The 4HV production was

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not dependent on ATP and required low aeration; all of these are considered beneficial

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characteristics for the production of target compounds, especially at an industrial scale.

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Under optimized conditions in a 5 L fermenter, the titer, productivity, and molar

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conversion efficiency for 4HV reached 100 g/L, 4.2 g/L/h, and 92%, respectively. Our

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system could prove to be a promising method for the large-scale production of 4HV

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from LA at low-cost and using a renewable biomass source.

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Keywords: 4-hydroxyvalerate; levulinic acid; 3-hydroxybutyrate dehydrogenase

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(3HBDH); formate dehydrogenase (FDH).

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1. INTRODUCTION Levulinic acid (LA) can be produced by a relatively simple acid hydrolysis of

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various cellulosic biomass1, 2. Several studies have focused on LA biorefineries as a

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novel option for efficient utilization of biomass because LA is a sustainable platform

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compound, which can be used for the synthesis of commercially valuable chemicals

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such as fuel additives, fragrances, solvents, oil additives, pharmaceuticals, and

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plasticizers3. The production of 4-hydroxyvalerate (4HV) from LA has attracted

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attention because 4HV can be used as a monomer for polyhydroxyalkanoates (PHAs)4, 5

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and as a precursor for gamma-valerolactone (GVL)6. These chemicals are widely used

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as a precursor of block copolymers7, 8, advanced fuel9, and acrylic compound for drug

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delivery10.

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Biological synthesis of 4HV from LA can avoid the problems incurred in chemical

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synthesis such as need of harsh conditions, expensive catalysts, organic solvents, and low

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yield11-13. Moreover, whole-cell biotransformation provide an excellent basis for efficient

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and sustainable catalysis because the metabolism of living cells can be used to

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regenerate cofactors and enzymes14. Biological synthesis of 4HV has been reported in

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Pseudomonas putida KT244011-13. A maximal yield of approximately 27 g/L of 4HV has

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been achieved with LA using the strain P. putida KT2440 overexpressing the E. coli

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tesB (acyl-CoA thioesterase II) with a conversion efficiency of 26%11, 12. Recently the

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pathway of LA catabolism, mediated by lva operon, was reported in P. putida

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KT244015. The first step is the conversion of LA to LA-CoA by LvaE, an ATP

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dependent CoA ligase, using two ATP molecules (ATP to AMP). LA-CoA is reduced to

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4HV-CoA by LvaD, which requires NAD(P)H as a reducing agent. Thereafter 4HV-CoA

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is phosphorylated to 4PV-CoA by LvaAB and consequently acetyl-CoA and propionyl3



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CoA are produced, which are incorporated into the tricarboxylic acid cycle (TCA). To

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harness the elucidated pathway, lvaAB was deleted from the lva operon, which resulted

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in the overproduction of 4HV up to 50 g/L; this increased the precursor 4HV-CoA pool

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in P. putida KT2440 overexpressing tesB13.

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Enzymatic conversion of LA to 4HV was performed using an engineered 3-

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hydroxybutyrate dehydrogenase (H144L/W187F) (hereafter referred as 3HBDH*) from

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Alcaligenes faecalis. The 3HBDH* was obtained by a rational design approach with

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molecular docking simulation such that its substrate specificity was altered from 3-

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hydroxybutyrate to LA16. The 3HBDH* showed high catalytic activity towards LA by

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utilizing NADH, whereas the wild-type enzyme did not exhibit any catalytic activity

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towards LA. Because of the reaction equilibrium of the enzymatic reaction (LA to

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4HV), the limited conversion efficiency of 57% was achieved by the 3HBDH* within

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24 h16. Another limitation of this in vitro system is the need for regeneration of NADH.

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However, compared to 4HV production by harnessing the LA catabolic pathway13, this

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enzymatic reaction has certain advantages such as fewer enzymatic reactions and no

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ATP requirement (Figure 1).

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The whole-cell biotransformation has been shown to be cost effective and does

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not require enzyme purification17. In this study, we focused on the production of 4HV

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from the renewable substrate LA in an E. coli strain by utilizing 3HBDH* by whole-cell

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biotransformation. In addition, a cofactor regeneration system catalyzed by an NAD+-

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dependent formate dehydrogenase (CbFDH) obtained from Candida boidinii was

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employed. CbFDH converts formate to CO2 and reduces NAD+ to NADH. This

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conversion is almost irreversible catalytic reaction that can drive unfavorable redox

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reactions, and formate and CO2 themselves have no influence on the activities of partner 4


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enzymes. We successfully decoupled 4HV production from ATP production and NADH

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regeneration through cellular metabolism by coexpressing 3HBDH* and CbFDH in E.

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coli. By optimizing the culture conditions, we achieved high titer, productivity, and

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molar conversion efficiency at high cell density culture.

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2. MATERIALS AND METHODS

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2.1. Bacterial strains and plasmids

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The strains and plasmids used in this study are listed in Table 1. E. coli DH10B

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was used for all the molecular cloning work. Escherichia coli MG1655 (MG) was used

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as the parental strain for 4HV production. The 3HBDH* gene was amplified from pET-

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22b (+)−3HBDH* (kindly gifted by Prof. Young Je Yoo)16, cloned into the NdeI and

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XhoI sites of pBbE6k_rfp18 to construct the plasmid pBbE6k_3HBDH*. Subsequently,

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pBbE6k_3HBDH* was transformed into MG by electroporation using a MicroPulser

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electroporator (Bio-Rad) to yield the strain MG-H. The codon optimized CbFDH gene

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was amplified from pET-23b (+)−CbFDH (kindly gifted by Prof. Yong Hwan Kim)19,

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cloned into the NdeI and XhoI sites of pBbB6a_gfp to construct the plasmid

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pBbB6a_CbFDH. Subsequently, pBbB6a_CbFDH was co-transformed with

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pBbE6k_3HBDH* into MG to yield the strain MG-HF.

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2.2. Culture conditions

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Whole-cell biotransformation was performed to produce 4HV from LA. A

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single colony of the recombinant E. coli strains- MG-H or MG-HF, was inoculated into

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5 mL of Luria−Bertani (LB) medium (10 g/L tryptone, 5 g/L yeast extract, and 10 g/L 5



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NaCl) supplemented with ampicillin (100 mg/L) and kanamycin (50 mg/L) and cultured

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overnight at 37 °C with shaking at 200 rpm. The overnight grown culture was

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transferred (1:100) into 400 mL of modified terrific broth medium (12 g/L tryptone, 24

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g/L yeast extract, 4 g/L glycerol, 2.3 g/L KH2PO4, and 12.5 g/L K2HPO4)20 and cultured

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at 37 °C with shaking at 200 rpm. The expression of the target genes was induced by the

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addition of 0.1 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) when the optical

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density at 600 nm (OD600) reached 0.5. After culturing cells at 37 °C for 20 h with

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shaking at 200 rpm, the cells were harvested by centrifugation (12000 ×g for 15 min at

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4 °C), washed twice with potassium phosphate buffer (0.1 M, pH 6), and used as

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biocatalysts in the biotransformation studies. LA was neutralized with 10 N NaOH,

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sterilized, and used as a substrate for biotransformation. Sodium formate was used as a

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co-substrate.

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2.3. Optimization of whole-cell biotransformation conditions

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The biotransformation conditions for MG-HF (expressing 3HBDH* and

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CbFDH), including pH, temperature, aeration, molar ratio of formate, and LA, were

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optimized in 250 mL shaker flasks with 20 mL potassium phosphate buffer (0.1 M, pH

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6) medium amended with 23.2 g/L (0.2 M) of LA and inoculated with 50 gcww/L of cells.

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The medium was supplemented with 0.1 mM of IPTG, 1X trace element solution (2.4

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g/L FeCl3·6H2O, 0.3 g/L CoCl2·H2O, 0.15 g/L CuCl2·2H2O, 0.3 g/L ZnCl2, 0.3 g/L

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Na2MO4·2H2O, 0.075 g/L H3BO3, and 0.495 g/L MnCl2·4H2O)21, and appropriate

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antibiotics. To optimize the conditions, a different range of each parameter was

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analyzed: temperature (25 to 65 °C), pH (3.5 to 8.5), aeration (25 to 200 rpm), and

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molar ratio of formate to LA (0.2 to 1.4). In pH, temperature, and aeration optimization

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studies, 0.2 M formate was added. The pH optimization studies were performed at 6


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37 °C. For the temperature optimization studies, the initial pH was kept at 6. Except the

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aeration dependence studies, all other biotransformations were carried out under

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microaerobic conditions (50 rpm). The samples were collected after 6 h of

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biotransformation to quantify the concentrations of 4HV, LA, and formate by high

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performance liquid chromatography (HPLC).

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2.4. Whole-cell biotransformation in mini-bioreactor

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Whole-cell biotransformation using the strain MG-HF was carried out in a

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homemade mini-bioreactor (300 mL) equipped with the precise monitoring and control

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systems for pH, temperature, and stirring speed. The biotransformation medium and

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conditions were as follows: 100 mL of potassium phosphate buffer (0.1 M, pH 5.5)

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medium supplemented with 80 g/L (0.68 M) of LA and 46.2 g/L (0.68 M) of sodium

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formate, 50 gcww/L of cells, 400 rpm, and 37 °C. The pH was maintained at 5.5

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throughout biotransformation by automatic addition of 5 N HCl. Samples were

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collected at every 6 h of biotransformation to quantify the product by HPLC.

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2.5. Production of 4HV by two stage pH-stat fed batch in a 5 L fermenter

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A 5 L fermenter (MARADO-PDA; CNS, Daejeon, Korea) equipped with a

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precise monitoring and controlling systems for pH, temperature, dissolved oxygen, anti-

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foaming, and stirring speed was used for the two-stage pH-stat fed batch cultivation and

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biotransformation. The overnight culture of the recombinant strain MG-HF, grown in

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the LB broth, was transferred (1:10) to 5 L fermenter containing 1.8 L of modified

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terrific broth. After reaching the OD600 of approximately 15, the cells were induced by

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the addition of 0.1 mM IPTG. During the cell growth phase, the agitation, aeration, pH,

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and temperature were maintained at 700 rpm, 0.5 vvm air, 7.0, and 37 °C, respectively. 7



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The pH was controlled by automatic addition of 23% NH4OH and 4 M H3PO4 in batch

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culture. In this study, a modified pH-stat fed batch cultivation was applied with two

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different feeding solutions (FS1 and FS2). When the pH increased to a higher value

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than the set value or fluctuated frequently (approximately after 4 h of cultivation),

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indicating the depletion of the principal carbon source, the pH-stat feeding was started

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with FS1 containing 700 g/L of glycerol and 19.6 g/L of MgSO 4. In pH-stat batch,

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oxygen was supplied in an appropriate amount to maintain the dissolved oxygen level at

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50. When the OD600 of the culture reached approximately 50 to 60, biotransformation

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was initiated by adding the substrate solution (200 mL) containing LA and formate (at a

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final concentration of 0.2 M each), and the agitation, aeration, pH, and temperature were

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maintained at 400 rpm, 0 vvm air, 6.0, and 37 °C, respectively. In the biotransformation

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phase, the FS2, containing 4 M of levulinic acid (not neutralized), 4 M of formate and

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0.4 M of glycerol, was fed in response to pH stat (pH 6). When the pH increased above

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6 because of the enzymatic reactions on sodium formate, FS2 was automatically fed to

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reduce the pH, which served as a substrate and co-substrate source as well. Samples

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were collected at different time points for 12 h from the biotransformation initiation

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phase for product quantification by HPLC.

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2.6. Analytical methods

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For OD measurements, the culture samples were diluted appropriately to an

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OD600 value less than 0.8 and the OD was measured using a spectrophotometer (Libra

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S22; Biochrom, Cambridge, UK). Residual LA, 4HV, formate, and glycerol

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concentrations were measured using HPLC (Shimadzu HPLC station equipped with a

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Shimadzu refractive index detector and a Shimadzu SIL-20A autosampler). Briefly, 1

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mL culture medium was collected and centrifuged (16000 ×g) at room temperature for 8


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15 min. The supernatant was heated at 80 °C for 1 h to denature the remaining soluble

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proteins and centrifuged (16000 ×g) for 30 min to remove the denatured proteins. The

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final supernatant was then diluted 10-fold and analyzed by HPLC. LA and 4HV were

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quantified using Zorbax SB-Aq column (Agilent) as described previously13. For formate

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and glycerol quantification, the samples were eluted through an Aminex HPX-87H

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column (Bio-Rad) using 25 mM sulfuric acid as the mobile phase (flow rate of 0.6

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mL/min). LA and sodium formate were purchased from Sigma-Aldrich (St. Louis, MO)

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and 4HV was prepared by saponification of GVL with 10 N NaOH4.

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3. RESULTS AND DISCUSSION

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3.1. Effects of NADH availability on 4HV production in E. coli expressing 3HBDH*

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MG-H expressing 3HBDH* was used for whole-cell biotransformation of 4HV.

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A maximum of approximately 0.42 g/L 4HV was produced after 9 h of

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biotransformation (data not shown). A possible reason for this low 4HV production by

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MG-H could be the limited reaction equilibrium of 3HBDH* oxidoreductase reactions

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coupled with the NADH/NAD+ conversion, resulting in the reversible reactions between

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LA and 4HV. NADH production under normal cell metabolism is thought to be

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insufficient for providing a strong driving force for NADH regeneration. Intracellular

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cofactor concentrations might affect the efficiency of whole-cell biocatalysts17, 22.

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NADH/NADPH-dependent oxidoreductase reactions can be improved with a suitable

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cofactor regeneration system23. Glucose dehydrogenase (GDH) and formate

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dehydrogenase (FDH) are widely used for cofactor regeneration24. NADH is formed

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when formate is converted to CO2 by FDH. Advantages of using FDH include the low-

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cost and safety substrate, easy removal of CO2, and absence of byproduct formation25. 9



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NADH synthesis is thermodynamically favorable. Moreover, synthesis of LA from

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cellulosic biomass concomitantly produces formate as a coproduct26, which is an

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additional advantage of using LA hydrolysate for 4HV production. Therefore, to

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increase the intracellular NADH pool, we combined FDH based NADH regeneration

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system with the energy-conserving pathway to produce 4HV by 3HBDH*.

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3. 2. Optimization of biotransformation conditions to increase 4HV production

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3. 2. 1. Effects of temperature on 4HV production

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To examine the effects of temperature on 4HV production, the

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biotransformation was conducted at 25 to 65 °C. The maximum production (10 g/L) of

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4HV was attained at 37 °C (Figure 2a). Though the optimal growth temperature of C.

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boidinii strain for FDH was 30 °C and A. faecalis strain for 3HBDH* was 20-37 °C27, 28,

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the maximum concentration of 4HV was obtained at 37 °C. Resting cultures of a wide

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range of microorganism frequently perform well at temperatures considerably higher

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than those for optimal growth29. The temperature that supported the best performance of

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the cell free-CbFDH was 37 °C30.

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3. 2. 2. Effects of pH on 4HV production

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To examine the effects of pH on 4HV production, biotransformation was

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performed with different initial pH (3.5 to 8.5 at 0.5 interval). pH 5.5 was found to be

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optimum for 4HV production by MG-HF. 4HV production increased sharply at pH 4.5,

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reaching the maximum production (11.3 g/L) at pH 5.5, and gradually decreased when

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pH increased over 5.5 (Figure 2b). The optimum pH (5.5) we obtained was very close to

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the previously reported optimum pH values for 3HBDH* and CbFDH16, 30, 31. A possible

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reason for the high production at a lower pH (5.5) than the physiological pH (around 10


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7.2) could be the enhanced diffusion of substrate into cells in its acidic form32. The pKa

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of LA (calculated by Chemicalize, an online platform. Source:

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https://chemicalize.com/#/calculation) shows that only 0.21% of LA is present in acid

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form at pH 7, and levels of LA in acid form gradually increased to 6.22% when pH was

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reduced to 5.5. 4HV was not produced when pH reduced to 4.5 or lower as it was

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beyond the working pH range of enzyme.

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3. 2. 3. Effects of ratio of formate to LA on the 4HV production

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In the present study, formate was used as a co-substrate to regenerate NADH. A

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range of molar ratios of formate to LA (0 to 1.4 at an interval of 0.2) was examined to

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determine the optimum ratio for high 4HV production. 4HV production increased with

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increasing formate concentration till the molar ratio exceeded 1.0 (Figure 2c). The 1: 1

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molar ratio was chosen as the optimum ratio for biotransformation. Addition of formate

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increased 4HV production by approximately 23-folds over that obtained in the control

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without formate. Cofactor supply could be a limitation if the biosynthesis rate is high33.

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In whole-cell biotransformations, the redox cofactors and other biomass precursors are

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not utilized for biomass synthesis by the resting cells34. Therefore, the regenerated

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NADH was efficiently used for 4HV production by 3HBDH*. CbFDH mediated

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conversion of formate to CO2 is an almost irreversible catalytic reaction, and formate

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and CO2 have not been shown to influence the catalytic activities 3HBDH*, the primary

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enzyme involved in the biotransformation.

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3. 2. 4. Effects of aeration on 4HV production

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The effects of aeration (dissolved oxygen) on 4HV production were determined by performing whole-cell biotransformation under different aeration conditions. 11



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Different aeration conditions were maintained to control concentration of dissolved

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oxygen by adjusting the rotation speed (25, 50, 75, 100, 150, and 200 rpm). 4HV

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production was high (10.7 g/L) at 50 rpm (Figure 2d), therefore 50 rpm was chosen as

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optimum shaking speed for resting cell biotransformation. In the case of at 25 rpm, 4HV

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production was slightly lowered than that of at 50 rpm, which might be due to the

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reduction of the cell surface area for the substrate because the cells clumped tightly at

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the bottom of the flask35. Moreover, 4HV production decreased when the shaking speed

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exceeded 50 rpm. The high production of 4HV at low rpm could be due to the

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accumulation of NADH in microaerophilic conditions (reducing environment)17.

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Samuel et al. (2017) obtained the maximum titer of 2,3-butanediol using an engineered

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Bacillus subtilis strain co-expressing CbFDH for NADH regeneration under

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microaerobic environment (50 rpm). In aerobic environment, NADH is oxidized by the

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electron transport system with oxygen as a final electron acceptor resulting in lower

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intracellular NADH levels36. Therefore, under anaerobic condition, the amount of

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NADH in the whole-cell biotransformation process is remarkably increased37. Figure 2d

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shows that microaerophilic condition is necessary to attain the maximum 4HV

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production from LA by the system we used. This is probably because the LA to 4HV

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biotransformation step does not require ATP, which is mainly produced in the presence

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of oxygen.

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3. 3. Whole-cell biotransformation in mini-bioreactor

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Whole-cell biotransformation has gained more attention than free enzymes for

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the production of biochemicals because it protects enzymes in harsh environments from

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external forces and does not require the addition of cofactors. In addition, inhibition of

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cell growth by the substrate is eliminated38. Whole-cell biotransformation was 12


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performed under optimized conditions in a 300 mL bioreactor with 100 mL of

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potassium phosphate buffer (0.1 M, pH 5.5) medium containing 50 gcww/L, LA (80 g/L),

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and sodium formate (46.2 g/L). After 24 h of cultivation, the system produced 40 g/L of

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4HV with a conversion efficiency of 86 % using the strain MG-HF (Figure 3).

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Approximately 51% higher conversion efficiency was achieved using whole-cell

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biotransformation (3HBDH*) with a cofactor regeneration system (CbFDH) than that

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using the in vitro bioconversion of LA by free enzyme 3HBDH* alone16. This result

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reiterates that whole-cell biotransformation using the NADH regeneration system is a

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more economical and efficient method than the free-enzyme system for the sustainable

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production of 4HV from LA.

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3.4. Two-stage fed-batch 4HV production in a 5 L fermenter

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High cell density can be used to increase the productivity39. However, cell

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enrichment for biotransformation on a large scale is a difficult process. To overcome

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this limitation, we attempted to produce 4HV with two-stage fed batch culture system

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without enrichment or washing process in a 5 L fermenter. High cell density cultivation

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(101 g/L E. coli with a specific growth rate of 0.1 h-1) was achieved by the pH-stat fed-

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batch culture, a cultivation strategy that couples nutrient feeding with pH

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measurement40. In the present study, the pH-stat fed-batch culture was used to attain the

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high cell density (OD600 of approximately 60) after 12 h of inoculation. The two-stage

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fed batch production was grouped into two phases—the growth phase and the

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production phase. In the growth phase, the strain MG-HF was grown for at least 12 h

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under pH stat, where OD600 reached approximately 60. Growth under pH-stat conditions

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has been reported as an efficient strategy to attain high cell density and produce high

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concentrations of various value-added chemicals in E. coli40-43. After 12 h of cultivation, 13



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the production phase was initiated by the addition of LA and formate. After 24 h of

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substrate addition, the strain MG-HF converted 330.8 g (2.85 mol) of LA to 303.3 g

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(2.57 mol) of 4HV, with 92% molar conversion efficiency. The titer, productivity, and

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molar conversion efficiency of 4HV from LA were 100.1 g/L, 4.2 g/L/h, and 92%,

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respectively. The titer, productivity, and molar yield increased by 2.5-fold, 2.6-fold, and

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1.1-fold, respectively, in a 5 L bioreactor compared with the biotransformation of LA in

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a 300 mL mini bioreactor (Figure 4). This indicates that energized cells are more

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advantageous for the whole-cell biotransformation than buffer-washed resting cells for

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the production of 4HV. E. coli has the advantage of rapid growth and potential for high-

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density cultivation44. Additionally, the theoretical density limit of E. coli liquid culture

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is approximately 1 × 1013 viable cells/mL (200 gcdw/L)42. Therefore, high-density cell

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cultivation was easily achieved. However, compared to E. coli, P. putida—that is widely

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used to produce 4HV from LA11-13—tends to have a lower growth rate in media such as

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Luria Bertani (LB) broth and minimal salt media45, 46. Therefore, E. coli is a more

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suitable host for the industrial production of 4HV than P. putida.

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Furthermore, the 4HV productivity obtained in the present study was

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considerably better (5- to 8-fold higher) than that obtained in the previous studies using

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P. putida KT2440 and harnessing the lva operon11, 13. One of the reasons for the

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increased 4HV production in the present study could be the requirement of less energy

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than that required for the lva operon based 4HV production in P. putida. The later

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process requires two ATPs and one NAD(P)H to produce one mole of 4HV from LA.

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Moreover, ATP is most efficiently supplied via oxidative phosphorylation. However,

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large fermenters potentially produce a local anaerobic or microaerobic environment

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during fermentation, which is not suitable for ATP-demanding bioconversions. In 14


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addition, vigorous aeration is required to efficiently utilize the ATP-demanding

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metabolic pathways and the aeration cost accounts for 26% of the utility cost47. When

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aeration is limited (30 vs 200 rpm), significantly higher levels of isopropanol were

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produced by the two-ATP-demanding pathway than the three-ATP-demanding

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pathway48. Thus, to improve the efficiency of the process and to reduce the aeration

330

cost, large-scale 4HV production is viable through the energy-conserving pathways,

331

such as 3HBDH* mediated conversion of LA to 4HV without ATP. The 4HV production

332

system presented here has the potential to produce 4HV from renewable feedstocks at a

333

commercial scale.

334

To conclude, high yield and productivity of 4HV were successfully achieved by

335

decoupling 4HV production from ATP production and NADH regeneration through

336

cellular metabolism by co-expressing 3HBDH* and CbFDH in E. coli. The system has

337

several advantages such as use of low cost substrate and co-substrate, no ATP

338

requirement, less aeration, cost effectiveness, high cell density cultivation, one pot

339

production with high titer, no production of byproducts, irreversible reactions catalyzed

340

by CbFDH, easy removal of the coproduct (CO2) as a gas from the medium, and no

341

interference with the purification of the final product. Therefore, the system has a

342

potential to produce 4HV, an important chemical, at industrial scale from the

343

renewable biomass.

344

345

FUNDING SOURCES

346

This work was supported by the Industrial Strategic Technology Development Program

347

(#10077308) funded by the Ministry of Trade, Industry & Energy (MOTIE, Korea), the 15



348

research program (NRF-2017R1A2B4003492) of National Research foundation of

349

Korea (NRF), and the Next-Generation BioGreen 21 Program funded by the Ministry of

350

Agriculture, Food, and Rural Affairs (SSAC, Grant No. PJ013457).

351

352

ACKNOWLEDGEMENTS

353

We thank Prof. Young Je Yoo of Seoul National University, Korea and Prof. Yong Hwan

354

Kim of UNIST, Korea for kindly providing the plasmids, pET-22b (+)−3HBDH* and

355

pET-23b (+)−CbFDH.

356

357

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358

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509

510

511

512 22


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513

Figure Captions

514

Figure 1.

515

indicates the acid hydrolysis of lignocellulosic biomass. Thick arrow indicates the

516

energy-conserving pathway to produce 4HV by the 3HBDH* using one NADH and the

517

CbFDH mediated-cofactor regeneration system. Thin arrows indicate the catalytic

518

conversions of LA into 4HV through engineering the lva operon and expressing tesB

519

using ATP and NAD(P)H.

520

Figure 2. Optimization of biotransformation conditions to increase 4HV

521

production. Effects of temperature (a), pH (b), ratio of formate to levulinate (c), and

522

aeration (d) on biotransformation of LA to 4HV by the strain MG-HF were determined.

523

Data show the production of 4HV after 6 h of biotransformation. Data represent the

524

means of three independent experiments and error bars represent standard deviation.

525

Figure 3. Production of 4HV in a mini-bioreactor by the strain MG-HF.

526

Equimolar concentrations of LA and formate were used and product concentration was

527

increased to approximately 40 g/L of 4HV with a conversion efficiency of 86%. Data

528

represent the means of three independent experiments and error bars represent standard

529

deviation.

530

Figure 4. Production of 4HV by the two-stage pH-stat fed batch in a 5 L fermenter.

531

Biotransformation was initiated at 12 h of cultivation with the addition of LA and

532

formate (at a final concentration of 0.2 M each). In the biotransformation phase, the

533

feeding solution (FS2), containing 4 M of LA, 4 M of formate and 0.4 M of glycerol,

534

was fed in response to pH stat. A maximum of 100 g/L of 4HV was achieved after 12 h

Metabolic pathways involved in the production of 4HV. Dotted arrow

23



535

of biotransformation. Data represent the means of three independent experiments and

536

error bars represent standard deviation.

537

538

539

540

541

542

543

544

545

546

547

548

549

550

551

552

553 24


Page 24 of 30

Page 25 of 30


Table 1. Strains and plasmids used in this study

Strains and plasmids

Genotype and description

References

MG1655

E. coli K-12 F–λ–ilvG–rfb-50rph-1

49

DH10B

F– mcrA Δ(mrr-hsdRMS-mcrBC) φ80lacZΔM15 ΔlacX74 recA1 endA1 araD139 Δ(ara-leu)7697 galU galK λ– rpsL(StrR) nupG

50

MG-H

MG1655 harboring pBbE6k-3HBDH*

This study

MG-HF

MG1655 harboring pBbE6k-3HBDH* and pBbB6a- CbFDH

This study

Strains

Plasmids pET-22b(+)−3HBDH*

pET-22b(+) with 3HBDH* (H144L/W187F)

16

pET-23b(+)−CbFDH

pET-23b(+) with CbFDH

19

pBbE6k_rfp

colE1 ori, carrying PLlacO-1 and rfp, KmR

18

pBbB6a_gfp

BBR1 ori, carrying PLlacO-1 and gfp, AmpR

18

pBbE6k_3HBDH*

pBbE6k_rfp with Δrfp::3HBDH*, KmR

This study

pBbB6a_CbFDH

pBbB6a_gfp with Δgfp::CbFDH, AmpR

This study

25



Figure 1.

Cellulosic biomass Acid hydrolysis De/re-hydration

LvaE

CO2

Levulinic acid (LA)

ATP, CoA

AMP

Levulinyl-CoA (LA-CoA) NAD(P)H

NADH

3HBDH*

CbFDH NAD

LvaD

+

+

NAD(P)

Formic acid TesB

4-hydroxyvaleric acid (4HV)

CoA

4-hydroxyvaleryl-CoA (4HV-CoA)

26


Page 26 of 30

Page 27 of 30


Figure 2. (b) 12

12

10

10 4HV (g/L)

4HV (g/L)

(a)

8 6

4 2

6 4 2

0

0 25

30

35

40 45 50 55 Temperature (°C)

60

65

(c)

3

4

5

6 pH

7

8

9

175

200

(d) 12

12

10

10 4HV (g/L)

4HV (g/L)

8

8 6 4 2

8 6 4 2

0

0 0

0.2 0.4 0.6 0.8 1 1.2 Molar ratio of formate:levulinate

1.4

25

27


50

75 100 125 150 Shaking speed (rpm)


Figure 3. 100

554

Concentration (g/L)

LA

Formate

4HV

80

555 60

556

40

20

557

0 0

4

8

12 16 Time (h)

20

558 24

28


Page 28 of 30

Page 29 of 30


Figure 4. Growth phase

4HV, LA, Formate and Gly (g/L), Growth (OD600), and conversion efficiency (%)

120

100

4HV LA Gly Formate Growth Conversion efficiency

pH-stat Fed-batch

Batch

559

Biotransformation phase

560 561

80

562 563

60

564 40

565 566

20

567 0 0

4

8

12

16

20

24

28

Time (h)

32

36 568

569

29



Page 30 of 30

Graphic for table of contents

Biomass

Chemical Precursor

Optimizing Bioconversion

Commercial Products

Solvents

Levulinic acid Formic acid Glucose Mannose Galactose Xylose Arabinose

One pot biotransformation

Drugs

100 g/L 4HV

Decoupled from cell metabolism

High cell density

30


Polymers

Fuel additives

Flavors

Personal care Agro-chemicals

High-level production of 4-hydroxyvalerate from levulinic acid

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