Engineering the lva operon and Optimization of Culture Conditions for

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Engineering the lva operon and optimization of culture conditions for enhanced production of 4-hydroxyvalerate from levulinic acid in Pseudomonas putida KT2440 Chandran Sathesh-Prabu, and Sung Kuk Lee J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b06884 • Publication Date (Web): 18 Feb 2019 Downloaded from http://pubs.acs.org on February 19, 2019

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Journal of Agricultural and Food Chemistry

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Engineering the lva operon and optimization of culture conditions for enhanced

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production of 4-hydroxyvalerate from levulinic acid in

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Pseudomonas putida KT2440

4 Chandran Sathesh-Prabua and Sung Kuk Leea,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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Department of Chemical Engineering, Ulsan National Institute of Science and

Department of Biomedical Engineering, Ulsan National Institute of Science and

Technology (UNIST), Ulsan 44919, Republic of Korea

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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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1 ACS Paragon Plus Environment


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Abstract

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Monomeric 4-hydroxyvalerate is a versatile chemical used to produce various

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commodities and fine chemicals. In the present study, the lvaAB gene was deleted from

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the lva operon in Pseudomonas putida KT2440 and the tesB, obtained from Escherichia

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coli, was overexpressed under the control of the lva operon system that is inducible by

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substrate levulinic acid and product 4-hydroxyvalerate to produce 4-hydroxyvalerate

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from levulinic acid. The lvaAB deleted strain showed almost complete conversion of

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levulinic acid to 4-hydroxyvalerate, compared with 24% conversion in the wild type

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strain. In addition, under optimized culture conditions, the final engineered strain

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produced a maximum of 50 g/L of 4-hydroxyvalerate with 97% conversion from levulinic

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acid. The system presented here could be applied to produce high titers of 4-

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hydroxyvalerate in a cost-effective manner at a large scale from renewable cellulosic

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biomass.

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Key words: 4-hydroxyvalerate; levulinic acid; Pseudomonas putida; TesB

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1. Introduction

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Hydroxy acids including 4-hydroxyvalerate (4HV) are versatile compounds that

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are used to produce various products such as polyesters, biodegradable and bio-

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compatible polyesters, fine chemicals, and pharmaceuticals because of their interesting

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reactivity patterns.1–4 More importantly, 4HV can be lactonized to yield γ-valerolactone

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(GVL) which can be used as fuels, solvents, food additives, and precursors of value-added

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carbon chemicals.5 In general, 4HV is produced for the synthesis of some intracellular

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polyhydroxyalkanoates (PHAs) by many types of bacteria.2,3,6–8 4HV contributed up to

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30 mol% of the hydroxyalkanoate constituents when 4-hydroxyvaleric acid was the sole

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carbon source.3 It provides better physical and mechanical properties to the biopolymers

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(polyesters) upon polymerization with other hydroxy acids.3,6

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Monomeric 4HV is obtained either via chemical synthesis as an intermediate9,10

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or via the depolymerization of intracellularly produced PHA4 or biological synthesis.1,11

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Both methods suffer from various shortcomings, including low yield, use of harsh

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conditions, catalysts, and organic solvents, need for chemical modification, and

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incomplete depolymerisation.1 The third method, biological synthesis, achieved high

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titers of 14 g/L and 27 g/L of 4HV from the renewable carbon source levulinic acid (LA)

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in shake flasks and bioreactors, respectively, using the Pseudomonas putida strain

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overexpressing the gene for a recombinant acyl-CoA thioesterase (TesB, for the removal

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of CoA carriers from hydroxy acids) obtained from Escherichia coli.1,11 In both studies,

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a maximum of around 25-26% molar conversion was obtained. In another study, 4HV

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was synthesized from LA in vitro using a variant enzyme, 3-hydroxybutyrate

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dehydrogenase (3HBDH), obtained from Alcaligenes faecalis, which achieved a 57%

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conversion within 24 h.12



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LA, a member of the gamma-keto acids, can be obtained through acid-catalyzed

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dehydration and the hydrolysis of sugars from renewable cellulosic feedstocks, such as

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rice hulls, rice straw, corn stalks, corn starch, cotton stem, sawdust, wheat straw, and

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shredded paper.13,14 The P. putida KT2440 strain, generally recognized as safe (GRAS)

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as a microbial host for recombinant DNA constructs, has become a next generation

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synthetic biology chassis or industrial workhorse thanks to its metabolic versatility and

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applicability.15,16 The strain exhibits excellent properties, including a remarkable

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tolerance to oxidative stress, organic solvents, and aromatic compounds, no or reduced

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by-product formation, and a high yield of NADPH through the Entner–Doudoroff (ED)

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pathway.15,17,18 Recently, the LA catabolism mediated by a seven-gene lva operon,

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encoded by lvaABCDEFG, has been elucidated in P. putida KT2440.19 In the LA

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assimilation pathway, LA is first activated as a coenzyme A-thioester, levulinyl-CoA

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(LA-CoA), by an acyl-CoA synthetase (LvaE) with an expense of ATP and CoA. Next,

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LvaD (short-chain dehydrogenase-reductase family) catalyzes the reduction of LA-CoA

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with either NADH or NADPH to yield 4-hydroxyvaleryl-CoA (4HV-CoA). Subsequently,

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4HV-CoA is phosphorylated to yield 4-phosphovaleryl-CoA (4PV-CoA) by the

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combined action of LvaA (phosphotransferase family) and LvaB (hypothetical protein)

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with an ATP. Then, the 4PV-CoA is converted into 3-hydroxyvaleryl-CoA (3HV-CoA)

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by LvaC (acyl-CoA dehydrogenase family), which can be further oxidized via β-

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oxidation to yield acetyl-CoA and propionyl-CoA or incorporated into PHA polymers.19

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In the present study, the LA assimilation pathway was directed to the 4HV-

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synthesis pathway by overexpressing E. coli thioesterase II (TesB) on an LA- and 4HV-

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inducible expression system for the removal of CoA carriers from 4HV-CoA to yield


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4HV. In addition, P. putida KT2440 was engineered to obtain high titers of 4HV with the

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highest conversion from the renewable feed stock, LA (Figure 1).

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2. Materials and methods

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2.1. Microbial strains and plasmids

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The P. putida KT2440 strain was used as the parental strain for all genetic

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modifications, including gene knockouts and the introduction of expression constructs.

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The E. coli strain DH10B was used for cloning. The constructed strains and plasmids are

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listed in Table 1.

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2.2. Chemicals, enzymes, and culture conditions

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All chemicals and media were purchased from Sigma-Aldrich (St. Louis, MO).

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Restriction enzymes (New England Biolabs, Ipswich, MA), DNA ligase (New England

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Biolabs, Ipswich, MA), and Pfu-X DNA polymerase (Solgent, Daejon, Republic of Korea)

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were used for cloning and plasmid construction. LA was neutralized with 10N NaOH and

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sterile-filtered prior to use. 4HV was prepared by alkaline saponification of GVL with 10

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N NaOH.3 The media, Luria-Bertani (composition per liter: 5 g yeast extract; 10 g peptone,

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and 10 g NaCl) and terrific broth (composition per liter: 12 g tryptone; 24 g yeast extract;

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9.4 g potassium phosphate, dibasic, and 2.2 g potassium phosphate, monobasic), were

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used to cultivate the E. coli and P. putida KT2440 at 37°C or 30°C under aerobic

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conditions, respectively, with agitation in an orbital incubator shaker at 200 rpm. The

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media were supplemented with 50 μg/mL of kanamycin (Km) where required.

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2.3. Strain and plasmid construction

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Primers used in this study are listed in Table S1. The engineered (deletion or

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insertion) P. putida KT2440 strains (Pp:ΔAB, Pp:ΔAB:ΔGlpR, and Pp:ΔAB:gTesB)



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were constructed by using a scar-less chromosomal in-frame gene deletion or insertion

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method based on the sacB negative counter-selection system employing the pQSAK

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plasmid (kindly gifted by Prof. Sunghoon Park), as previously described.20 Briefly, to

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delete lvaAB, glpR or tesB, a fragment containing the ~500 bp upstream and downstream

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regions of the target gene was PCR-amplified and cloned into the pQSAK. Subsequently,

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the constructed plasmid was transformed into electro-competent cells of the respective

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strains by electroporation using a MicroPulser electroporator (Bio-Rad) to yield the

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strains Pp:ΔAB, Pp:ΔAB:ΔGlpR and Pp:ΔAB:ΔGlpR:ΔTesB by a double homologous

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recombination. The electro-competent cells were prepared as previously described.21 For

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the insertion of tesB, the PCR-amplified tesB of E. coli was flanked by ~500 bp of the

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upstream and downstream regions of the lvaAB gene using overlap extension PCR. Then,

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the product was cloned into the pQSAK plasmid and the tesB chromosome integrated

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P. putida KT 2440 strain (Pp: ΔAB:gTesB) was constructed as described above.

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To construct the LA inducible expression system through the LvaR, the fragment

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(lvaR-PlvaA), containing LvaR and the intergenic region between the LvaR-coding

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sequence and the translational start site of the lva operon, was generated by PCR using

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P. putida KT2440 genomic DNA. Then, the segment was cloned into the Broad-host-

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range promoterless probe vector, pPROBE-gfp, to yield the pPROBE_LvaR_PlvaA_GFP

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plasmid. The constructed plasmid was transformed into the electro-competent cells of

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Pp:ΔAB to obtain the strain Pp:ΔAB:PlvaA:GFP. The induction of PlvaA through LvaR was

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analyzed by estimating the fluorescence intensity of Pp:ΔAB:PlvaA:GFP cultivated in the

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respective medium supplemented either with LA or 4HV (each 10 mM). For 4HV

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production, the fragment (lvaR-PlvaA) was cloned into the pPROBE-TesB to express TesB

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under the control of LvaR_PlvaA. The constructed plasmid, pPROBE_LvaR_PlvaA_TesB,


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was transformed into WT, Pp:ΔAB, and Pp:ΔAB:ΔGlpR to yield the engineered strains

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Pp:ΔAB:TesB and Pp:ΔAB:ΔGlpR:TesB, respectively. All constructs were confirmed by

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DNA sequencing (Macrogen, Korea).

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2.4. Efficiency of the constructed systems

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2.4.1. Effect of the lvaAB deletion on cell growth on levulinic acid

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The growth of the lvaAB deleted strain (Pp:ΔAB) and the WT was analyzed on

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M9 medium (pH 7.0). A M9 medium containing the following components per liter was

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used: 6.78 g sodium phosphate, dibasic; 3 g potassium phosphate, monobasic; 0.5 g

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sodium chloride; 1 g ammonium chloride; 2 mM magnesium sulfate, and 0.1 mM calcium

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chloride. The medium was supplemented with 20 mM of LA as a sole carbon source. Cell

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growth was recorded by measuring absorption at 600 nm using a microplate reader

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(Infinite F200 PRO, Tecan) in a Corning 96-well, clear bottom plate with shaking (200

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rpm, 30°C). Furthermore, the consumption of LA by Pp:ΔAB and WT was analyzed by

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culturing in 20 mL TB medium supplemented with 0.4% glycerol and 10 g/L of LA.

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2.4.2. Culture optimization and production of 4-hydroxy valerate from levulinic

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acid

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The optimization of culture conditions included the following parameters: (i) the

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selection of medium (LB or TB); (ii) the selection of co-substrate (glucose or glycerol);

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(iii) the optimum concentration of glycerol and (iv) the maximum concentration of

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substrate (feeding). The engineered strains grown overnight in LB broth were added to

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the either 20 mL of fresh LB or TB media in 250 mL flask at an initial optical density

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(OD600) of 0.05 and were incubated at 30°C in a shaking incubator (200 rpm). The LB or

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TB media was amended with 4 mM magnesium sulfate, 0.2 mM calcium chloride, and

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0.1 mg/liter ferric ammonium citrate.11 As a co-substrate, either glucose (0.4%) or



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different concentrations of glycerol (0.4%, 0.8% or 1.6%) were added to the culture

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medium. To determine the substrate feeding rate, the LA (10, 15, 20 or 25 g/L) from the

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40% stock solution was added to the culture medium when the culture OD600 reached

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around 1.0-1.5. The culture was then incubated at 32°C for the better activity of TesB1.

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After the first addition of LA, the LA was added batch-wise (each batch containing 10,

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15, 20 or 25 g/L) to the cultures every 24 h of cultivation for up to 48 h or 72 h. Samples

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were collected at different time points for OD600 and HPLC analysis.

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2.5. Analytical and statistical determinations

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Cell density was monitored by absorbance at 600 nm (OD600) using a Biochrom

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Libra S22 spectrophotometer (Biochrom, Cambridge, UK). HPLC analysis [Shimadzu

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HPLC station (Shimadzu, Kyoto, Japan) equipped with a refractive index detector

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(Shimadzu) and a SIL-20A auto-sampler (Shimadzu)] was performed for the

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quantification of LA, 4HV, and glycerol in the cell-free supernatant from the culture

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media of recombinant P. putida KT2440 strains. For LA and 4HV quantification, the

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samples were eluted through a 4.6 mm × 150 mm, 5 μm Zorbax SB-Aq column (Agilent,

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USA) at 40°C using 25 mM ammonium formate (pH 2.0) as the mobile phase with a flow

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rate of 1 mL/min. For glycerol quantification, the samples were eluted through a 300 mm

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× 7.8 mm Aminex HPX-87H column (Bio-Rad, USA) at 40°C using 5 mM sulphuric acid

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as the mobile phase with a flow rate of 0.6 mL/min. Substrate conversion efficiency was

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calculated on a molar basis. All experimental data were subjected to one-way analysis of

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variance (ANOVA) or multivariate analysis of variance (MANOVA) using SPSS

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(Version 11) software (SPSS Inc., Chicago, IL) to determine the level of significance.

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P values < 0.05 were considered significant.

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3. Results and Discussion

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3.1. Effect of lvaAB deletion on cell growth and 4-hydroxyvalerate production from

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levulinic acid

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LA can be used as a sole carbon source through the LA assimilation pathway19

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and can be converted into PHA through the PHA-synthesis pathway3 in P. putida KT2440.

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The synthesis of our desired product (4HV) can be enhanced by engineering the lva

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operon to increase precursor availability (4HV-CoA). To achieve this, the 4HV-CoA

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consuming pathway was blocked by deleting the lvaAB genes in the lva operon of P.

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putida KT2440. Here, we evaluated the growth of the wild type (WT) and LvaAB-deleted

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strain (Pp:ΔAB) on a M9 medium supplemented with 20 mM of LA as a sole carbon

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source (Figure 2A) on a microtiter plate reader. As expected, the Pp:ΔAB strain did not

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grow on LA, but WT did (Figure 2A), suggesting that the deletion of lvaAB affects the

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LA catabolism by blocking the conversion of 4HV-CoA into central metabolites by the

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subsequent reactions to provide biomass and energy. It is worth noting that no homologs

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to lvaAB are active in P. putida KT2440.

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Furthermore, LA consumption in the Pp:ΔAB strain was compared with WT when

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cultivated in TB medium amended with LA (Figure 2B). The results show that even after

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48 h of cultivation, around 75% of the amended LA remained in the culture medium of

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Pp:ΔAB, whereas WT utilized almost all the LA, reflecting the cell growth. In an attempt

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to produce 4HV-containing polyesters, Gorenflo and co-workers3 found that only 44.5%

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of the metabolized LA was converted by the cells to the desired product, whereas 55.5%

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was presumably metabolized to CO2, cell mass, and other products in P. putida GPp104.

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Interestingly, Pp:ΔAB produced 2.4 g/L (accounting for 25% of the amended LA) of 4HV,

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suggesting that P. putida KT2440 has short-chain or broad chain-length specific



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thioesterases to transform 4HV-CoA into 4HV. LA was not catabolized by other

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unfavorable routes into central metabolites in the Pp:ΔAB strain, which is considered one

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of the most favorable traits of an industrial strain to achieve a high titer and yield of a

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desired product. Therefore, the engineered strain Pp:ΔAB was used for further study.

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3.2. Levulinic acid- and 4-hydroxyvalerate-inducible gene expression using lvaR-

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PlvaA system

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Rand and co-workers19 demonstrated that the lva operon is upregulated by a

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transcriptional activator, LvaR. Here, we employed this LvaR protein to construct an LA

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inducible system. The segment (lvaR-PlvaA), containing LvaR and the intergenic region

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between the LvaR-coding sequence and the translational start site of the lva operon, was

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linked upstream of GFP in pPROBE-gfp. The induction of the PlvaA through LvaR was

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analyzed by estimating the fluorescence intensity of the strain, Pp:ΔAB:PlvaA:GFP,

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(harboring pPROBE_LvaR_ PlvaA_GFP) cultivated in LB medium supplemented either

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with LA or 4HV (each 10 mM). It was observed that the strain Pp:ΔAB:PlvaA:GFP induced

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with LA showed a significantly higher fluorescence intensity compared to that of the

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control condition (Figure 3A). Moreover, the system was also found to be induced by the

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addition of 4HV. The findings are in accordance with a recent study.19 However, in the

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present study, LA showed higher efficiency over 4HV. The reason for this observation

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will have to be elucidated by further study. Since LB is a rich medium containing various

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components that could influence the induction of fluorescence, the fluorescence intensity

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assay was also carried out in the M9 medium supplemented with glucose and different

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concentrations of LA or 4HV. The results clearly show that LA and 4HV can induce the

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expression system (Figure 3B-3C) even at the lowest concentration (1 mM) of LA or 4HV.

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The system was found to also be induced by other short chain organic acids such as acetate,


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propionate, butyrate, valerate, and hexanoate. However, the induction efficiency was not

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comparable with that of LA or 4HV.19

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Substrate and/or product inducible systems are considered efficient systems in any

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field whose aim is the biosynthesis of target compounds, especially at industrial scales.

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The system constructed here could solve the limitations encountered with inducible or

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constitutive expression systems22,23 and offers an interesting phenomenon: the system can

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be induced by both the substrate and the product. Thanks to its low cost, LA can be used

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as a starting material to produce a wide range of chemicals.13,24

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3.3. 4-hydroxyvalerate production in the lvaAB deleted strain

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Previously, it was reported that 4HV can be produced from LA by overexpressing

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TesB in P. putida KT2440.1,11 In the present study, TesB from E. coli was overexpressed

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under the control of PlvaA to remove CoA from 4HV-CoA to yield 4HV. The efficiency

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of the present system was evaluated by estimating the 4HV titers in the WT:TesB and

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Pp:ΔAB:TesB strains (Figure 4). The strains were grown in 20 mL TB medium amended

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with 10 g/L of LA in a 250 mL- shake flask. LA (10 g/L) was added to the cultures after

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3 h of cultivation (OD600 = 1.0-1.5). Figure 4 shows that the strain Pp:ΔAB:TesB

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produced almost triple the amount (4.6 g/L of 4HV) of the 4HV when compared to that

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of the WT:TesB. The Pp:ΔAB:TesB strain resulted in a conversion of around 99%, over

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four-fold higher than that of the WT:TesB strain (24%). It has been reported that 4HV

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production requires a rich medium, and the use of LB medium with glucose was found to

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result in a superior production of 4HV than M9 medium.1 Furthermore, the TB medium

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with glucose was used in a 2 L bioreactor-fed-batch process to produce high titers of 4HV

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(27 g/L) and GVL (8 g/L) from LA.11 Therefore, TB was used to produce HV from LA

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in shake flasks. It is worth noting that around 55% of the LA amended still remained in



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the TB culture of the Pp:ΔAB:TesB strain even after 24 h of cultivation. Since the lvaAB

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was deleted, the production of other by-products such as 3HV from 3HV-CoA was also

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inhibited. During the production of 4HV from LA, around 3 g/L of 3HV (corresponding

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to a yield of 7% from LA) was obtained.1 As such, the advantages of the lvaAB deletion

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include the blocking of the complete catabolism of LA into the central metabolites and

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the blocking of the production of other by-products. The HV production pathway (LA →

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LA-CoA→ 4HV-CoA → 4HV) consumes at least two ATP and one reducing equivalent

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(NAD(P)H) to produce 4HV.19 Therefore, TB medium was supplemented with an

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additional carbon source, such as glucose or glycerol.

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3.4. Optimization of culture conditions

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Here, different concentrations of glucose or glycerol were added to the TB

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medium with higher concentrations of LA (20 g/L) to produce 4HV. LA was added to the

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cultures in two batches (each 10 g/L), after 3 h and 24 h of cultivation. The TB medium

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(a two-fold higher 4HV titer) performed better than the LB medium even without an

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additional carbon source. This suggests that the pathway demands a much richer medium.

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Figure 5A shows that without any additional carbon sources, both the LB and TB media

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produced significantly lower 4HV than their counterparts. However, the strain

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Pp:ΔAB:TesB yielded around 7 g/L and 11 g/L of 4HV from the TB medium

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supplemented with either glucose or glycerol (each 0.4%), respectively, after 48 h of

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cultivation. The concentration of glycerol was further increased to 0.8% and 1.6%. When

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the TB medium was supplemented with 1.6% glycerol, Pp:ΔAB:TesB significantly

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produced the highest amount of 4HV, around 19 g/L from 20 g/L of LA after 48 h of

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cultivation. It was found that the addition of glycerol increased the titer by 57% more than

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glucose. A possible reason for this increased titer could be the production of higher


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amounts of ATP and NAD(P)H, which are cofactors of 4HV production. Glycerol has a

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higher degree of reduction than glucose and produces twice as much reducing equivalents,

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when converted to phosphoenolpyruvate.25,26

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Since the lvaAB deleted strain showed the better conversion efficiency, efforts

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were undertaken to overproduce 4HV by adding higher concentrations of LA and glycerol

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to the TB medium. When producing 4HV with 20 g/L of LA and 1.6% of glycerol (this

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combination yielded the highest titer (Figure 5A)), around 0.25% of glycerol remained in

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the medium after 48 h of cultivation (data not shown). Therefore, LA and glycerol were

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added to the TB medium in a ratio of 1:0.68. The strain was grown in TB medium and

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different concentrations of LA were added batch-wise (each batch containing 15, 20, or

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25 g/L) to the cultures after 3 h and at every 24 h of cultivation for up to 48 h or 72 h,

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such that concentrations were kept low in the medium. The conditions were as follows:

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LA 15x3 (15 g/L LA added at 3, 24, and 48 h); LA 15x4 (15 g/L LA added at 3, 24, 48,

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and 72 h); LA 20x3 (20 g/L LA added at 3, 24, and 48 h), and LA 25x3 (25 g/L LA added

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at 3, 24, and 48 h). The 4HV tiers of LA 15x3, LA 15x4, LA 20x3, and LA 25x3 were 44

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g/L, 47 g/L, 38 g/L, and 37 g/L, respectively (Figure 5B). These results suggest that there

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was a reduction in 4HV titer when the total concentration of LA exceeded 60 g/L (LA

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25x3) or when a batch-wise concentration exceeded 15 g/L (LA 20x3 and LA 25x3).

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When the LA concentration was increased over 15 or 20 g/L, growth was retarded (Figure

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S1). Moreover, even in the fed-batch system with a 2 L bioreactor, LA was maintained

309

around 20 g/L to avoid toxicity.11 Figure 5C depicts the growth pattern of Pp:ΔAB:TesB

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while grown under the four conditions (LA 15x3, LA 15x4, LA 20x3, and LA 25x3).

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There was no growth defect in LA 15x3 and LA 15x4, however, growth was affected in

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LA 20x3 and LA 25x3, possibly due to high concentrations of LA.



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3.5. Increase in 4-hydroxy valerate production by deletion of glpR gene

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Despite the richness of the TB medium used to produce 4HV from LA, the highest

315

titer was only obtained after the addition of glycerol to the culture. The P. putida KT2440

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strain cultured with glycerol as the sole carbon source displayed a prolonged lag phase

317

(>10 h).27,28 This long-lag phase was eliminated by the deletion of glpR (encoding the

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glycerol-3-phosphate-responsive transcriptional repressor), which controls the expression

319

of the glpFKRD gene cluster encoding the enzymes needed for glycerol catabolism.28

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In the present study, the glpR gene was deleted in the Pp:ΔAB:TesB strain to

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obtain the strain Pp:ΔAB:ΔGlpR:TesB. Furthermore, TesB was also integrated into the

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chromosome of the Pp:ΔAB strain, under the control of PlvaA, to obtain the strain

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Pp:ΔAB:gTesB. Both strains (Pp:ΔAB:ΔGlpR:TesB and Pp:ΔAB:gTesB) were evaluated

324

for the production of 4HV from LA under the conditions as LA 15x4 and were compared

325

to the production of the Pp:ΔAB and Pp:ΔAB:TesB strains (Figure 6A).

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Pp:ΔAB:ΔGlpR:TesB slightly increased 4HV production, from 47 g/L to 50 g/L,

327

compared to that of Pp:ΔAB:TesB. The glpR deletion in Pp:ΔAB:TesB positively

328

influenced 4HV production. It has been reported that the lva operon operates under carbon

329

catabolite repression (CCR) mediated by glucose.19 In the present study, glycerol and LA

330

were simultaneously utilized by the engineered strain, suggesting that no CCR was

331

exerted by glycerol (Figure S2). Figure 6b shows various parameters, including the

332

addition and consumption of LA, the production of 4HV, cell growth, and molar

333

conversion, in the biosynthesis of 4HV by Pp:ΔAB:ΔGlpR:TesB under the conditions of

334

LA 15x4. The chromatogram of the produced 4HV was compared with the standard

335

(Figure S3). The production of 4HV was at least 1.9-fold higher with a 4-fold higher

336

conversion (%) in this strain compared to that of the fed-batch system in TB medium


Page 14 of 30

Page 15 of 30


337

supplemented with 2% glucose in 2 L bioreactor,11 even without addition of any external

338

inducer. On the other hand, 4HV production of Pp:ΔAB:gTesB (TesB on chromosome)

339

was at least 3.5-fold lower than that of Pp:ΔAB:TesB (TesB on plasmid). However, its

340

4HV production was 1.4-fold higher than that of Pp:ΔAB. In Pp:ΔAB, the endogenous

341

thioesterases produced around 10 g/L of 4HV (a 20% of the total 4HV produced). To

342

confirm the involvement of the endogenous TesB (PP_4762) on the production of 4HV,

343

the endogenous tesB was deleted from Pp:ΔAB:ΔGlpR to yield the strain

344

Pp:ΔAB:ΔGlpR:ΔTesB. The resulting strain showed reduced 4HV production of 3 g/L,

345

as compared with 12 g/L of 4HV produced by Pp:ΔAB:ΔGlpR cultured under the

346

conditions of LA 15x4 (data not shown). Figure 6A shows that the expression and activity

347

of TesB is essential for the production of 4HV.

348

To conclude, we employed an LA inducible expression system to produce 50 g/L

349

4HV with 97% conversion by P. putida KT2440. To our knowledge, this is the highest

350

production of the industrially important monomeric hydroxy acid, 4HV, in a P. putida

351

strain from LA, a renewable, low-cost feedstock. An increased conversion rate (%) was

352

obtained by the engineered strain which could not completely catabolize the substrate or

353

intermediates into the central metabolites. The reported system offers several advantages,

354

including no catabolite repression, no requirement of external inducers, no production of

355

by-products, cost-effectiveness, and industrial scale-up suitability.

356 357

FUNDING SOURCES

358

This work was supported by the industrial strategic technology development

359

program (#10077308) by the Ministry of Trade, Industry & Energy (MOTIE, Korea) and



360

the Next-Generation BioGreen 21 Program funded by the Ministry of Agriculture, Food

361

and Rural Affairs (SSAC, Grant No. PJ01345701).

362 363

ACKNOWLEDGEMENT

364 365

We thank Prof. Sunghoon Park of UNIST, Korea for kindly providing the plasmid pQSAK.

366 367

REFERENCES

368

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glycerol in Escherichia coli. Biotechnol. Bioeng. 2009, 103, 148–161.

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V. The glycerol-dependent metabolic persistence of Pseudomonas putida KT2440

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463 464 465 466 467 468 469 470 471 472 473 474 475 476 477 478 479


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480


Figure captions

481 482

Figure 1. Schematic representation of various strategies applied to engineer P. putida

483

KT 2440 for enhanced production of 4HV from LA. lvaAB gene from the lva operon

484

was deleted. Escherichia coli tesB was overexpressed under the control of the lva operon

485

system in a LA- and 4HV-inducible expression system. glpR was deleted to improve

486

glycerol catabolism. The readers are referred to the text for more details.

487 488

Figure 2. Effect of lvaAB deletion on cell growth and 4HV production from LA. (A)

489

Effect of lvaAB deletion on the growth of Pp:ΔAB and WT on M9 medium supplemented

490

with 20 mM of LA as the sole carbon source. Growth was measured on a microtiter plate

491

reader. (B) Consumption of LA by Pp:ΔAB and WT in TB medium amended with 10 g/L

492

of LA. Data represent the mean of three different experiments, and error bars represent

493

standard deviation.

494 495

Figure 3. LA- and 4HV-inducible gene expression using lvaR-PlvaA system.

496

Fluorescence intensity (GFP) was measured in Pp:ΔAB:PlvaA:GFP by culturing in LB

497

medium with 10 mM of LA or 4HV (A) or in M9 medium with different concentrations

498

of LA (B) or 4HV (C). Data represent the mean of three different experiments, and error

499

bars represent standard deviation.

500 501

Figure 4. 4HV production in the lvaAB deleted strain. 4HV production was evaluated

502

in WT:TesB and Pp:ΔAB:TesB by culturing in TB medium amended with 10 g/L of LA.

503

Pp:ΔAB:TesB yielded a conversion of around 99%, which is more than four-fold higher



504

than that of WT:TesB (24%). LA was added at 3h of cultivation. Data represent the mean

505

of three different experiments, and error bars represent standard deviation.

506 507

Figure 5. Optimization of culture conditions. (A) 4HV was produced by Pp:ΔAB:TesB

508

in different media. TB medium supplemented with 1.6% glycerol was found to be the

509

optimum combination to attain the highest concentration of 4HV. (B) 4HV production by

510

Pp:ΔAB:TesB with increased concentrations of both LA and glycerol. LA and glycerol

511

were added into the TB medium in a ratio of 1:0.68. (C) The growth pattern of

512

Pp:ΔAB:TesB while culturing under four conditions (LA 15x3, LA 15x4, LA 20x3, and

513

LA 25x3). Data represent the mean of three different experiments, and error bars represent

514

standard deviation. Glu, Glucose; Gly, Glycerol.

515 516

Figure 6. Increase in 4HV production by deletion of glpR gene. (A) Production of

517

4HV by different engineered P. putida KT2440 strains. Pp:ΔAB:ΔGlpR:TesB,

518

Pp:ΔAB:gTesB, Pp:ΔAB:TesB, and Pp:ΔAB were evaluated for the production of 4HV

519

from LA under the conditions of LA 15x4. (B) Detailed parameters of the biosynthesis of

520

4HV by Pp:ΔAB:ΔGlpR:TesB. Data represent the mean of three different experiments,

521

and error bars represent standard deviation.

522 523 524 525 526


Page 22 of 30

Page 23 of 30

527


Table 1. Strains and plasmids used in this study Strains and plasmids

Description

Reference

Strains E. coli DH10B

Cloning host

Lab stock

WT

P. putida KT2440

Lab stock

Pp:ΔAB

P. putida KT2440 with ∆lvaAB

This study

Pp:ΔAB:PlvaA:GFP

Pp:ΔAB harbouring PlvaA-GFP

pPROBE-LvaR-

This study

WT:TesB

WT harbouring pPROBE-LvaR-PlvaATesB

This study

Pp:ΔAB:TesB

Pp:ΔAB harbouring PlvaA-TesB

This study

Pp:ΔGlpR

WT with ∆glpR

This study

Pp:ΔAB:ΔGlpR

Pp:ΔAB with ∆glpR

This study

Pp:ΔAB:ΔGlpR:TesB

Pp:ΔAB:ΔGlpR harbouring pPROBELvaR-PlvaA-TesB

This study

Pp:ΔAB:gTesB

Pp:ΔAB::TesB

This study

pPROBE-LvaR-

Pp:ΔAB:ΔGlpR:ΔTesB Pp:ΔAB:ΔGlpR with ΔtesB

This study

Plasmids pPROBE-gfp

pBBR1-ori, KmR, expression vector

pQSAK

ColE1-ori; sacB, KmR and AmpR

pPROBE-TesB

pPROBE-gfp with ∆gfp::TesB

Lab stock

pQSAK-lvaAB

Used to delete lvaAB in WT

This study

pQSAK-glpR

Used to delete glpR in WT and Pp:ΔAB

This study

pQSAK-tesB

Used to delete tesB in Pp:ΔAB:ΔGlpR

This study

pQSAK-gTesB

Used to integrate tesB in Pp:ΔAB

This study

pPROBE-LvaR-PlvaAGFP

lvaR-PlvaA cloned into pPROBE-gfp

This study

pPROBE-LvaR-PlvaATesB

broad-host

range

29 20

This study pPROBE-gfp with ∆gfp::lvaR-PlvaA-TesB



Page 24 of 30

Figure 1.

Production of 4HV under optimized conditions BBR1

Plasmid Kan

pPROBE

Engineered P. putida KT2440

4HV TesB

LA−CoA

LA ATP, CoA

LvaD

LvaAB

4HV−CoA

NAD(P)H

4PV−CoA

LvaC

β-oxidation

3HV−CoA

ATP

Chromosome lvaR

Biomass and Energy Transport and catabolism

LvaE

TesB

LvaR

lvaABCDEFG glpR

Glycerol

Advantages: • Substrate-induced gene expression

Commercial applications

• No catabolite repression • High production

50 g/L of 4HV

• Production from sustainable biomass TB+Gly+LA


Page 25 of 30


Figure 2.

A

1.2 1

OD600

0.8 0.6 0.4 0.2 0 0

4

8

12

16

20

Time (h) Pp:ΔAB

12

24

10 18

8 6

12

4

OD600

B

Con. of LA or 4HV (g/L)

WT

6 2 0

0 0

10

20

30

40

50

Time (h) LA consumption by Pp:ΔAB 4HV production by Pp:ΔAB Cell growth of Pp:ΔAB

LA consumption by WT 4HV production by WT Cell growth of WT



A

Fluorescence intensity

Figure 3.

3000

LB LB + 4HV LB + LA

2500 2000 1500 1000 500 0 0

4

8

12

16

20

12

16

20

12

16

20

Time (h)


B

3000

LA 0 mM LA 1 LA 2 LA 4 LA 6 LA 8 LA 10

2500 2000 1500 1000 500 0 0

4

8

C


Time (h)

3000

HV 0 mM HV 1 HV 2 HV 4 HV 6 HV 8 HV 10

2500 2000 1500 1000 500 0 0

4

8

Time (h)


Page 26 of 30

Page 27 of 30


12

100

10

80

8

60

6 40

4

Conversion (%)

Con. of LA or 4HV (g/L)

Figure 4.

20

2 0

0 0

6

12 Time (h)

18

24

4HV production by ΔlvaAB:TesB

4HV production by WT:TesB LA consumption by WT:TesB

LA consumption by ΔlvaAB:TesB

Molar conversion by WT:TesB

Molar conversion by ΔlvaAB:TesB



Figure 5.

A Con. of 4HV (g/L)

20

LB TB TB with Glu (0.4%) TB with Gly (0.4%) TB with Gly (0.8%) TB with Gly (1.6%)

15 10 5 0 0

B Con. of 4HV (g/L)

50

10

20 30 Time (h)

40

50

40

80

100

80

100

LA 15x3 LA 15x4

40

LA 20x3 LA 25x3

30 20 10 0

0

20

60

Time (h)

C

24

LA 15x3 LA 15x4

OD600

18

LA 20x3 LA 25x3

12 6 0 0

20

40

60

Time (h)


Page 28 of 30

Page 29 of 30


Figure 6.

A Con. of 4HV (g/L)

60

Pp:ΔAB Pp:ΔAB:gTesB

50

Pp:ΔAB:TesB

40

Pp:ΔAB:ΔGlpR:TesB

30 20 10 0 0

20

40

60

80

100

60

100

4HV

LA Growth Glycerol Conversion

50 40

80 60

30 40 20 20

10 0

Conversion (%)

B

Con. of LA, 4HV or Gly (g/L) or OD600

Time (h)

0

0

20

40

60

80

100

Time (h)



Page 30 of 30

Table of Contents Graphic (TOC graphic)

Pseudomonas putida KT2440

Chemical treatment

LA

LA

4HV

Lignocellulosic biomass Commercial utilization: Polymer precursor Solvents, fuel additives

4HV

Advanced biofuels Culture optimization


Engineering the lva operon and Optimization of Culture Conditions for

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