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Optimum rebalancing of the 3-hydroxypropionic acid production pathway from glycerol in Escherichia coli Hyun Gyu Lim, Myung Hyun Noh, Jun Hong Jeong, Sung Hoon Park, and Gyoo Yeol Jung ACS Synth. Biol., Just Accepted Manuscript • DOI: 10.1021/acssynbio.5b00303 • Publication Date (Web): 07 Apr 2016 Downloaded from http://pubs.acs.org on April 9, 2016

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ACS Synthetic Biology

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Optimum rebalancing of the 3-hydroxypropionic acid production pathway

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from glycerol in Escherichia coli

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Hyun Gyu Lima, Myung Hyun Noha, Jun Hong Jeonga, Sunghoon Parkb, *, Gyoo Yeol

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Junga,c,*

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a

Department of Chemical Engineering and cSchool of Interdisciplinary Bioscience and

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Bioengineering, Pohang University of Science and Technology, 77 Cheongam-Ro, Nam-Gu,

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Pohang, Gyeongbuk 37673, Korea

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b

School of Chemical and Biomolecular Engineering, Pusan National University, 2 Busandaehak-ro 63beon-gil, Geumjeong-gu, Busan 46241, Korea

13 *

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To whom correspondence should be addressed.

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(Sunghoon Park)

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Mailing address: School of Chemical and Biomolecular Engineering, Pusan National

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University, 2 Busandaehak-ro 63beon-gil, Geumjeong-gu, Busan 46241, Korea

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Tel.: +82-51-510-3049, Fax: +82- 51-515-2716, E-mail: [email protected]

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(Gyoo Yeol Jung)

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Mailing address: Department of Chemical Engineering, Pohang University of Science and

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Technology, 77 Cheongam-Ro, Nam-Gu, Pohang, Gyeongbuk 37673, Korea

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Tel.: +82-54-279-2391, Fax: +82-54-279-5528, E-mail: [email protected]

ACS Paragon Plus Environment


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Abstract

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3-Hydroxypropionic acid (3-HP) can be biologically produced from glycerol by two

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consecutive enzymatic reactions, dehydration of glycerol to 3-hydroxypropionaldehyde (3-

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HPA) and oxidation of 3-HPA. The pathway has been proved efficient, but imbalance

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between the rates of the two enzymatic reactions often results in the accumulation of the toxic

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3-HPA, which severely reduces cell viability and 3-HP production. In this study, we used

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UTR engineering to maximally increase the activities of glycerol dehydratase (GDHt) and

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aldehyde dehydrogenase (ALDH) for the high conversion of glycerol to 3-HP. Thereafter, the

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activity of GDHt was precisely controlled to avoid the accumulation of 3-HPA by varying

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expression of dhaB1, a gene encoding a main subunit of GDHt. The optimally balanced E.

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coli HGL_DBK4 showed a substantially enhanced 3-HP titer and productivity compared with

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the parental strain. The yield on glycerol, 0.97 g 3-HP/g glycerol, in a fed-batch experiment,

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was the highest ever reported.

16 17 18 19

Keywords

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3-hydroxypropionic acid, synthetic biology, metabolic engineering, balancing enzyme

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activity

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1. Introduction

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3-Hydroxypropionic acid (3-HP), which can be converted into many chemicals such

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as acrylic acid, acrylamide, and propiolactone, is an important platform chemical.1, 2 In 2004,

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the US Department of Energy (DOE) listed 3-HP as one of the top value-added chemicals

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produced from biomass.3 The global market of acrylic acid alone is expected to reach $18

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billion in 2020 (data from Allied Market Research) and this reflects the importance of 3-HP

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in industry. However, the chemical synthesis of 3-HP is not feasible due to high cost of the

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starting materials, generation of toxic intermediates, and/or other environmental issues.4

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Consequently, biological production of 3-HP has received much attention. Several microorganisms, including Klebsiella pneumoniae, Lactobacillus sp., and

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Chloroflexus aurantiacus, can naturally produce 3-HP as an intermediate or end product.1, 5

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From the early 2000s, several recombinant strains of Escherichia coli have been developed

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for the production of 3-HP with heterologous expression of relevant genes derived from the

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native 3-HP producers and other microorganisms. One such strain, expressing the coenzyme

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B12-dependent glycerol dehydratase (GDHt) and NAD+-dependent aldehyde dehydrogenase

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(ALDH) of K. pneumoniae, could produce 0.58 g/L 3-HP during growth on glycerol, which is

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the main byproduct of the biodiesel industry.6 Another E. coli strain, which harbors the B12-

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independent malonyl-CoA reductase from C. aurantiacus,7 could also produce 0.19 g/L of 3-

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HP from glucose. More recently, it was reported that β-alanine-pyruvate aminotransferase

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and 3-hydroxypropionate dehydrogenase could be employed to produce 3-HP from β-alanine

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via malonic semialdehyde.8, 9

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In spite of its dependence on coenzyme B12, 3-HP production by glycerol conversion

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using GDHt and ALDH is attractive because it is a relatively simple 2-step reaction, and

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gives higher titer and productivity than other biosynthetic pathways. To improve the 3-HP



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production from glycerol, many efforts were made towards both strain development

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(screening and engineering of pathway enzymes, modification of glycerol metabolism, etc.)

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and process development (culture condition optimization, high cell density culture, etc.).10-13

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However, the imbalance between the two reactions, i.e., dehydration of glycerol and the

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subsequent oxidation of 3-hydroxypropionaldehyde (3-HPA) to 3-HP, still remains a major

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issue.1 Once the imbalance occurs, the toxic 3-HPA accumulates and cell growth and 3-HP

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production are seriously hampered.14 This 3-HPA severely decreases cell growth and enzyme

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activities even at < 10 mM.15, 16 Although the exact mechanisms have not been elucidated, it

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was suggested that 3-HPA can cause DNA damage or react with the thiol group of cysteine to

10 11

ruin the structural integrity at the protein level.15, 17 Over the last few decades, the strategies to control gene expression for balancing

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biosynthetic pathway, including the use of promoters/ribosome binding site (RBS) with

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varying strengths, and plasmids with different copy number, have been adopted to control

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metabolic fluxes in vivo.18, 19 Combined strategies have greatly contributed to the prevention

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of accumulation of toxic intermediates and minimizing metabolic burden caused by excessive

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and non-optimal production of pathway enzymes during product formation.18 These strategies

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have proved to be successful in the production of terpenoids,20 taxadiene,21 amorphadiene,22

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and fatty acids.23 However, the control of gene expression is limited by the number of genetic

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elements; its further modification based on random library requires a time-consuming

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screening process.24 Recently, an in silico design tool called ‘UTR Designer’ has been

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developed for the precise prediction of the translation initiation efficiency. The prediction is

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based on the folding structure of the 5′ untranslated region (5′ UTR) of mRNA, which is

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affected by the 5′ proximal coding sequence of the structural gene, thus enabling the optimal

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design of the 5′ UTR region regardless of the coding sequences of the structural gene.25 This

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UTR Designer can be effectively utilized to intentionally control the expression of target


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genes for broad dynamic range.24, 25 This has already been applied for pathway

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amplification,26-29 co-factor balancing,30, 31 enzyme stabilization,32 and carbon flux

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redistribution.33

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In this study, UTR engineering was applied for the balanced amplification of the

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glycerol-dependent 3-HP production pathway. As E. coli DUBGK strain accumulated 3-

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HPA in the previous study,10 this strain was chosen as a parental strain for rebalancing.

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Expression of ALDH was maintained at a fixed high level, while that of GDHt was varied

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across a wide range. Because GDHt is a dimer of heterotrimers (α2β2γ2) and its α subunit is

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known to provide the substrate binding pocket,34 the α subunit was chosen for expression

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control under different 5′ UTRs, which were designed by the UTR Designer. The

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rebalancing of 3-HP pathway enabled us to develop an E. coli strain that produced 40.51 g/L

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3-HP with a high productivity (1.35 g/L/h) and yield (0.97 g 3-HP/g glycerol) without

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significant accumulation of 3-HPA. To the best of our knowledge, the yield of 3-HP on

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glycerol reported here is the highest thus far.

15 16



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2. Results and Discussion

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2.1 Pathway optimization strategy

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3-HP is produced from glycerol through a 2-step reaction, which is catalyzed by

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GDHt and ALDH (Fig. 1). In this study, GDHt, which is a dimer of the αβγ heterotrimer and

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encoded by dhaB1, dhaB2, and dhaB3, was derived from K. pneumoniae. ALDH, encoded by

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a single gene kgsadh, was derived from Azospirillum brasilense. During the reaction, GDHt

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and ALDH are inactivated by the toxic intermediate, 3-HPA. In the previous study,10 the

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accumulation of 3-HPA was suppressed by the expression of GDHt in the moderate copy-

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number pDK7 plasmid (p15A origin), and ALDH in the high copy-number pUC plasmid

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(pMB1 origin). Although the recombinant E. coli W DUBGK could produce > 40 g/L of 3-

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HP in 48 h, 3-HPA was still observed in early phase of the bioreactor experiments.10 After its

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accumulation, the activities of both GDHt and ALDH significantly decreased with time.10

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This indicated further pathway rebalancing of 3-HP biosynthetic pathway is required for

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higher production.

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To rebalance the pathway, we hypothesized that the accumulation of 3-HPA can be

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prevented by increasing the ALDH activity to a higher level while decreasing the GDHt

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activity to a lower level, compared to those of DUBGK. However, because the optimal

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activity ratio of the two enzymes is not known, we decided to start with the highest

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expression of both the GDHt and ALDH and gradually decrease the GDHt activity. To this

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end, the strong inducible tac promoter was employed for both operons expressing GDHt and

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ALDH. Moreover, the optimized 5′ UTRs for the maximum expression (Table 1) were

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employed for both enzymes. On the other hand, the high-copy pUC plasmid was used for

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ALDH and the medium-copy pACYC plasmid was used for GDHt, similar to the previously


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studied E. coli W DUBGK.10 In order to vary the GDHt activity later, dhaB1 was

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independently placed from dhaB2 and dhaB3 (Fig. 1). The other two genes encoding the

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reactivase of GDHt, gdrAB, were placed after dhaB3 to form a single polycistronic operon

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along with dhaB2 and dhaB3. In addition, acid tolerant E. coli W strain was also used as it is

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known to be more suitable for high production of various acids including 3-HP.10

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2.2 Pathway amplification by strong promoter and 5′ UTRs

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Before varying the expression level of dhaB1, performance of the strain with the

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highest expression for both GDHt and ALDH was examined. The two recombinant plasmids

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were introduced into the E. coli W strain35 to yield strain HGL_BK1. Enzyme activity of

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crude cell lysate of the resultant strain HGL_BK1 was measured and compared with that of

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the previously studied strain DUBGK. The activities of both GDHt (Fig. 2A) and ALDH (Fig.

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2B) increased by 27.3 % and 26.5%, respectively, indicating successful construction of

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recombinant plasmids and increased expression of the two enzymes by UTR engineering.

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The strain DUBGK and HGL_BK1 was cultivated in the same modified M9 minimal

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medium for 3-HP production. Upon addition of 0.1 mM isopropyl-β-D-thiogalactopyranoside

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(IPTG) and 2 µM coenzyme B12 to the culture medium, both strains immediately began to

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produce 3-HP. However, 3-HP production at 24 h was as low as 1.82 g/L (Fig. 2D), which

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was 30.5% less than that of DUBGK (Fig. 2C) constructed in a previous study.10 The initial

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growth rate and glycerol uptake rate up to 6 h of HGL_BK1 were also lower than those of

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DUBGK (16.3% and 36.1% less, respectively). On the other hand, HGL_BK1 accumulated

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less acetate (0.41 g/L) and 1,3-propanediol (PDO) (0.01 g/L) than the strain DUBGK (0.96

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g/L acetate and 0.07 g/L 1,3-PDO). HGL_BK1, not DUBGK, accumulated some 3-HPA at 6

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h. Overall, these results suggest that the newly constructed HGL_BK1 strain highly expressed



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both GDHt and ALDH, but not at optimal levels. The non-optimal overexpression of

2

biosynthetic enzymes was not beneficial for both cell growth and 3-HP production, and this is

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attributed to the metabolic burden and accumulation of 3-HPA.

4 5

2.3 Optimization of 3-HP production pathway by fine-tuning the expression of glycerol

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dehydratase

7 8

For fine-tuning of the catalytic activity for GDHt, the expression level of dhaB1 was

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varied by 5′ UTRs having different strengths (Table 1). The resulting recombinant plasmids

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and pUC_K plasmid (harboring kgsadh) were co-introduced to the E. coli W strain to yield

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the strains HGL_BK2–4 (Table 2). A negative control (HGL_BK5) was also constructed by

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introducing a blank plasmid. SDS-PAGE analysis on HGL_BK1–5 indicated that the

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expression of DhaB1 varied in accordance to the predicted expression level (Fig. 3A). To

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examine the effect of the varied expression of dhaB1 on 3-HP production, these strains were

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cultivated in the M9 medium containing 100 mM glycerol (Fig. 3B). Interestingly, 3-HP

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production generally increased as dhaB1 expression decreased. Strain HGL_BK4, which had

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the lowest expression of dhaB1 among the constructed recombinants, showed the highest 3-

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HP titer (4.40 g/L) after 24 h cultivation. Moreover, the productivity was also high in this

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strain at 0.18 g/L/h, which is 242% and 168% higher than that of strain HGL_BK1 and

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DUBGK, respectively. Furthermore, the strain HGL_BK4 produced negligible amounts of

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byproducts (0.54 g/L of acetate and 0.04 g/L 1,3-PDO), even without deleting the byproduct

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formation pathways (Fig. 3C). These results suggest that in HGL_BK4, the 3-HP biosynthetic

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pathway has been well established and the glycerol flux was mostly directed to 3-HP

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production.


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Enzyme activity of GDHt in the crude cell extract was also analyzed for HGL_BK 1–5

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strains (Fig. 3D). We could observe that the GDHt activity decreased when a weaker 5′ UTR

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was employed. Although the expression levels of dhaB1 with dhaB1-V3 and dhaB1-V4

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UTRs were indistinguishable on SDS-PAGE, the enzyme activity was different in accordance

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to the predicted UTR strength. This strong correlation with predicted expression level

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indicates that UTR engineering is an efficient and highly precise tool for translation control.

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Interestingly, the amounts of dhaB1 transcript were also influenced by UTR engineering (Fig.

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3E). While the amounts of dhaB1 transcript in HGL_BK1 and HGL_BK2 were similar, those

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in HGL_BK3 and HGL_BK4 were lower than two others. Presumably, active translation with

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high initiation rate could prevent cleavage by ribonuclease, thus resulted in increased stability

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of mRNA.36, 37

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Cell growth curve of the five variants until 9 h is also shown (Fig. 3F). All variants

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expressing GDHt and ALDH (HGL_BK1 to HGL_BK4) showed lower cell growth than the

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one with blank plasmid (HGL_BK5), suggesting that expression of 3-HP synthetic pathway

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is a significant metabolic burden to the host E. coli W. In addition, the initial cell growth of

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the variants decreased as the GDHt activity increased. The more reduced growth rate of the

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strain HGL_BK1 with higher GDHt activity is attributed to the higher accumulation of 3-

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HPA, 14, 16 although the level is not fully differentiable or even undetectable by HPLC.

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The strain HGL_BK4 was cultivated with increased glycerol concentration to

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examine its ability for 3-HP production more in details. When glycerol was added

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intermittently during cultivation, the HGL_BK4 strain produced 16.7 g/L 3-HP (Fig. 4A).

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However, 1.96 g/L acetate and 0.25 g/L 1,3-PDO also accumulated (Fig. 4B). To minimize

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the byproduct formation, ackA-pta and yqhD were deleted (Fig. 1), which were responsible

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for the synthesis of acetate and 1,3-PDO, respectively.13 Strain HGL_DBK4 (strain

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HGL_BK4 with ∆ackA-pta ∆yqhD), showed decreased production of acetate (1.48 g/L; 24.3%



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reduction) and 1,3-PDO (0.04 g/L; 86.0% reduction) in the 48 h cultivation (Fig. 4C). 3-HP

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production was improved to 17.9 g/L with a productivity of 0.37 g/L/h and a yield of 0.61 g/g,

3

respectively.

4 5

2.4 Fed-batch cultivation of the engineered strain

6 7

Fed-batch fermentation was carried out to fully elucidate the 3-HP production

8

potential of the stain HGL_DBK4 (Fig. 5). To obtain a high cell mass, both glycerol and

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glucose were intermittently added as carbon sources, but the concentration of glycerol in the

10

medium was maintained at less than 10 g/L. Following the induction with 0.1 mM IPTG,

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strain HGL_DBK4 produced 3-HP with consumption of both glycerol and glucose. The

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initial productivity was low as 0.30 g/L/h, but gradually increased to the maximum

13

production rate of 3.2 g/L/h after 15 h. At 30 h, the strain produced 40.51g/L 3-HP by

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consuming 41.79 g/L glycerol and 34.27 g/L glucose. The overall 3-HP productivity (1.35

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g/L/h) was significantly higher than the strain DUBGK (0.86 g/L/h).10 Moreover, the strain

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HGL_DBK4 produced marginal amount of byproducts (only 1.46 g/L acetate and 0.06 g/L

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1,3-PDO). Balanced amplification of the 3-HP synthetic pathway along with the deletion of

18

the byproduct formation pathways largely reduced the loss of carbon substrates to wasteful

19

products. The 3-HP production yield reached 0.97 g/g glycerol or 0.53 g/g total carbon source,

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which is the highest until date. Our results clearly demonstrate that the UTR engineering is an

21

efficient tool for balancing the 3-HP biosynthetic pathway.

22 23

2.5 Discussion

24


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Pathway amplification to accelerate and improve the carbon flux toward product

2

biosynthesis is a common practice in metabolic engineering. However, the engineered strains

3

often show unsatisfactory performance because of imbalance in metabolic fluxes. This

4

implies that the optimization of biosynthetic pathway is highly required to develop an

5

efficient cell factory. Balanced amplification of pathway mainly not to accumulate toxic

6

intermediates is an essential part for optimization; these can be achieved by the precise

7

control of gene expression of metabolic enzymes. In this manner, the UTR engineering can

8

be used to pathway optimization with an accurate design of predictable expression cassettes

9

for target genes. We demonstrated that the catalytic activity of GDHt was precisely controlled

10

to pre-determined levels by simply introducing a tailored 5′ UTR sequence in front of its

11

coding sequence. With the given 5′ UTR sequences and expression levels, amount of the

12

protein DhaB1 (from strong to nearly invisible expression on SDS-PAGE, Fig. 3A) and the

13

activity of GDHt (from 100% to 11%, Fig. 3D) was varied successfully.

14

Previously, several transcriptional control strategies were applied to regulate the

15

expression of single or multiple genes, and many successful results were reported.20-23

16

However, in many cases, cellular phenotype was not accurately regulated, due to lack of the

17

control over the translation process. As translation initiation is strongly affected by the

18

folding structure of mRNA in the 5′ UTR region and initial coding sequence of a structural

19

gene,25 a mere transcriptional control without a cautious consideration on the translation

20

initiation has had limitation. Additionally, combinatorial approaches by randomization of

21

promoter sequences (including UTR) and some of the initial codons of structural gene have

22

also been investigated to optimize gene expression.38-40 One serious drawback with the

23

combinatorial approach is that the library size can easily exceed the experimentally testable

24

number even with few bases; for example, with only 5 bases, the size is 45 which is larger

25

than 1,000. Another problem is that the library can be easily biased.24 The current UTR



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1

engineering can be combined with the purely combinatorial approach to reduce the library

2

size and its biased nature. It is possible to prepare a much smaller, but predictive

3

combinatorial library that has a uniform distribution between maximum and minimum with a

4

few modifications on the 5′ UTR by computational methods.24, 41 The optimal expression of

5

target genes can be less laboriously found by testing the affordable small library even without

6

the aim of screening tool.42, 43

7

In summary, we tried to rebalance the 3-HP biosynthetic pathway from glycerol to

8

develop 3-HP high producer through this work. We initially increased the activities of GDHt

9

and ALDH to their maximal levels with a strong inducible promoter and highly efficient 5′

10

UTRs. The amplification of the catalytic activity was successful; however, the non-optimal

11

overexpression of GDHt resulted in the accumulation of toxic 3-HPA, poor cellular growth,

12

and low 3-HP production. To rebalance the production pathway, we controlled the GDHt

13

activity by fine-tuning the expression of dhaB1, the major subunit of GDHt, with different-

14

strength 5′ UTRs. This significantly improved the productivity and yield of 3-HP, and we

15

finally concluded that a successful pathway rebalancing was achieved. The strategies and

16

optimally rebalanced pathway could be utilized for economic 3-HP production as a platform

17

in bio-refinery industry.

18 19


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3. Methods

2 3

3.1 Reagents, plasmids and strains

4 5

The strains used in this study are listed in Table 2. The plasmid DNA was isolated

6

using the AccuPrepR Nano-Plus Plasmid Mini Extraction Kit (Bioneer, Daejeon, Korea). The

7

oligonucleotides (Table 3) were synthesized by Cosmogenetech (Seoul, Korea). DNA

8

fragments amplified during PCR were purified using the GeneAllR ExpinTM Gel SV kit

9

(GeneAll, Seoul, Korea). The Phusion polymerase and restriction enzymes were obtained

10

from New England Biolabs (Ipswich, MA, USA), the EmeraldAmp GT PCR Master Mix

11

(Clontech Laboratories, Mountain View, CA, USA) was used for routine cloning procedures,

12

and reagents for cell cultivation were obtained from BD Bioscience (Sparks, MD, USA).

13

Other reagents were obtained from Sigma (St. Louis, MO, USA).

14

The pUC/K plasmid was prepared from pUC/KGSADH by replacing the parental

15

expression cassette with the synthetic expression cassette containing the tac promoter and the

16

5′ UTR designed for maximum expression. Plasmid segment amplified using the primer pair

17

pUC-F/pUC-B and pUC/KGSADH as the template, and an insert segment amplified using

18

the primer pair O-kgsadh-F1/F2/B and the same template were digested using SacI and

19

HindIII endonucleases, and ligated for construction of the pUC/K plasmid. To construct

20

pACYC_B0, a vector segment amplified using the primer pair pACYC-F/pACYC-B and

21

pACYC_duet as the template, and the insert segment amplified using the primer pair O-

22

dhaB2-F1/F2/B and pDK7 (p15A) _DhaB123, gdrAB as the template were digested using the

23

SpeI and PacI enzymes, and ligated. For cloning of dhaB1 with the designed 5′ UTR, the

24

fragments amplified using the O-dhaB1-F1/O-dhaB2-F2/O-dhaB1-B1/BO-dhaB1-B2 primer

25

set and pDK7 (p15A) _DhaB123, gdrAB were inserted at the SpeI restriction site in the



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

1

pACYC_B0 plasmid. The other variants of dhaB1 were cloned using the same procedure

2

except that the primers O-dhaB1-F1-V2, V3, and V4 were used instead of O-dhaB1-F1.

3

The 5′ UTR sequences were generated by UTR Designer, freely available via a

4

website (http://sbi.postech.ac.kr/utr_designer). To obtain 5’ UTR sequences with targeted

5

expression level, ‘N11~13AAGGAGN6~8’ and 35 nucleotides of each coding sequence were

6

uploaded as constraints for designing 5′ UTR. For maximum expression, desired expression

7

level was set to 6 million; this value was sequentially lowered when lower expression of gene

8

is required. The generated sequences were used for dhaB1, dhaB2, and kgsadh (Table 1).

9

Chromosomal manipulation for deleting the byproduct formation pathway (yqhD,

10

ackA-pta) was conducted using the Red recombination44, 45 with pKD46, pCP20, and sets of

11

primers listed in Table 3.

12 13

3.2 Media and culture conditions

14 15

The modified glycerol minimal medium used for flask-scale cultivation contained 0.5

16

g/L MgSO4.7H2O, 2 g/L NH4Cl, 2 g/L NaCl, 1 g/L yeast extract and 100 mM potassium

17

phosphate buffer (pH 7.0); 100 mM or 400 mM glycerol was included as the carbon source.

18

To maintain the plasmids, 50 µg/ml of kanamycin and 25 µg/ml of chloramphenicol were

19

added. The cells were cultured in 300 ml flasks containing 20 ml of medium, and incubated at

20

37°C with shaking (250 rpm). When 400 mM of glycerol was used, the pH was adjusted to 7

21

using 10 M NaOH solution every 6 h.

22

To test for 3-HP production, the seed culture was prepared by inoculating a colony

23

into Luria broth (LB) containing appropriate antibiotics. Following overnight growth, the

24

seed culture was refreshed by inoculating into the new medium with optical density (OD600)


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1

0.05. The cells were induced at OD600 0.8 by the addition of 0.1 mM of IPTG. Coenzyme B12

2

(2 µM) was added at 3 h and 12 h. The culture was conducted with three biological replicates.

3 4

3.3 Analysis of glycerol, 3-HP, and other metabolites

5 6

The concentration of glycerol, 3-HP, and other metabolites including 3-HPA,

7

acetate and 1,3-propanediol (1,3-PDO) were quantified at 14°C using a UltiMateTM 3000

8

analytical HPLC system (Dionex, Sunnyvale, CA, USA) and an HPX-87H column (Bio-Rad

9

Laboratories; Richmond, CA, USA) with 5 mM H2SO4 as the mobile phase at a flow rate of

10

0.6 ml/min. The signals were monitored using a Shodex RI-101 refractive index detector

11

(Shodex, Klokkerfaldet, Denmark).

12 13

3.4 Preparation of crude cell lysate

14 15

To prepare crude cell lysate for SDS-PAGE analysis and enzyme activity assays, the

16

cells were inoculated from the plates into LB with appropriate antibiotics. The overnight

17

seeds were refreshed, and 0.1 mM of IPTG was added when the OD600 was 0.6 - 0.8. The cell

18

pellets were harvested after 3 h. The cell lysate was prepared by addition of Bug Buster

19

Master Mix (EMD Bioscience, San Diego, CA, USA) to the cell pellet, and incubated in an

20

anaerobic chamber at room temperature for assay when the cells were fully lysed.

21 22

3.5 SDS-PAGE for expression analysis

23 24 25

For analysis of the expression of glycerol dehydratase, NuPAGE NovexR 4 - 12% Bis-Tris Gel (Life Technology; Waltham, MA, USA) was used following the manufacturer’s



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1

instructions. The protein samples were mixed with the same volume of SDS gel loading

2

buffer containing 100 mM Tris-Cl (pH 6.8), 4% (w/v) sodium dodecyl sulfate (SDS), 0.2%

3

(w/v) bromophenol blue, 20% (v/v) glycerol, and 100 mM dithiothreitol (DTT). The samples

4

were denatured by incubation at 70°C for 10 min. The gel was run at 200 V for 35 min using

5

NuPAGE MES SDS Running Buffer (Life Technology), and stained using a solution of 0.1%

6

Coomassie Brilliant Blue G-250 (w/v), 45% methanol (v/v), and 10% acetic acid (v/v).

7

Subsequently, the gel was destained using the staining solution without added Coomassie

8

Brilliant Blue. SeeBlueR Plus2 Pre-Stained Standard (Invitrogen, Waltham, MA, USA) was

9

used as a protein size marker.

10 11

3.6 Enzyme assay for glycerol dehydratase and aldehyde dehydrogenase

12 13

The activity of glycerol dehydratase was measured using a coupled reaction with

14

aldehyde hydrogenase from Saccharomyces cerevisiae (Sigma, St. Louis, MO, USA). This

15

involved monitoring the absorbance at 340 nm to detect an increase in the reduced form of

16

nicotinamide adenine dinucleotide (NADH) using a VICTOR3 1420 Multilabel Plate Reader

17

(PerkinElmer, Waltham, MA, USA) at 25°C. Because of the suicidal inhibitory effect of

18

glycerol on the enzyme,46 we used 1,2-propanediol as the substrate. Specifically, to initiate

19

the reaction, the lysate was mixed with reaction buffer containing 35 mM potassium

20

phosphate (pH 8.0), 50 mM potassium chloride, 50 mM 1,2-propanediol, 2 mM NAD+, 1 mM

21

DTT, 15 uM coenzyme B12, and 10 units/ml of aldehyde dehydrogenase.

22

In case of aldehyde dehydrogenase encoded in kgsadh, the method was basically

23

same with the procedure for glycerol dehydratase except use of the reaction buffer containing

24

35 mM potassium phosphate (pH 8.0), 50 mM potassium chloride, 5 mM propionic aldehyde,

25

2 mM NAD+.


Page 16 of 38

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1


The concentration of protein was calculated by the Bradford assay using Bio-Rad

2

Protein Assay Dye (Bio-Rad, Hercules, CA, USA), with bovine serum albumin as the

3

standard. The activity was normalized against the total protein concentration in the lysate to

4

obtain the specific glycerol dehydratase activity.

5 6

3.7 Quantification of the amount of dhaB1 transcript

7 8

The total RNA was extracted from each variant incubated for 2 h after induction with

9

0.1 mM IPTG using RNeasy Mini Kits (Qiagen GmbH, Germany). The samples were treated

10

with recombinant Dnase I (Takara Bio Inc., Kusatsu, Japan) to degrade residual DNA

11

according to manufacturer’s protocol. SuperScriptR III Reverse Transcriptase (Invitrogen)

12

and TOPrealTM qPCR 2X PreMIX (Enzynomics, Daejeon, Korea) was used for cDNA

13

synthesis and qPCR, respectively. The relative amount of dhaB1 transcript was quantified

14

using the comparative CT method (∆∆Ct) using rrsA as a reference.47

15 16

3.8 Bioreactor study

17 18

The recombinant strain was cultured at 37°C in a 5-L bioreactor containing a 2 L

19

initial working volume. The overnight seed culture was inoculated into a medium containing

20

100 mM potassium phosphate buffer, 2 g/L NaCl, 1 g/L yeast extract, 10 ml/L trace metal

21

solution,48 and 0.1% (v/v) antifoam 204. Sterile air was provided at a flow rate of 1 vvm, and

22

the culture was stirred at 500 rpm. The pH was adjusted to 7 with ammonia solution

23

(NH4OH). The glycerol and glucose was added intermittently. To produce 3-HP, IPTG was

24

added when the OD600 reached 1.0, and coenzyme B12 was added to a final concentration of



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1

20 µM at 3-h intervals. The concentrations of the carbon source and metabolites were

2

monitored periodically using HPLC.

3 4


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1 2


Author Information Corresponding Authors

3

For S. P., Tel.: +82-51-510-2395, Fax: +82- 51-515-2716, E-mail: [email protected]

4

For G.Y.J., Tel.: +82-54-279-2391, Fax: +82-54-279-5528, E-mail: [email protected]

5 6

Author Contributions

7

H.G.L., S.P., and G.Y.J. conceived the project. H.G.L. designed and performed

8

experiments, analyzed the data, together with M.H.N. and J.H.J. H.G.L., S.P., and G.Y.J.

9

conducted data analysis and interpretation and wrote the manuscript. S.P. and G.Y.J.

10

supervised the project. All authors read and approved the final manuscript.

11

12 13

Competing Interests The authors declare that they have no competing interests.

14 15

Acknowledgements

16

This research was supported by grants from the Advanced Biomass R&D Center

17

(ABC) of Global Frontier Project funded by the Ministry of Science, ICT and Future

18

Planning (ABC-2015M3A6A2066119); the Marine Biomaterials Research Center of the

19

Marine Biotechnology Program; and the Gyeongbuk Sea Grant Program, funded by the

20

Ministry of Oceans and Fisheries, Korea. We thank Dr. Sang Woo Seo for the critical

21

comments on the manuscript.

22



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1

Figure captions

2 3

Fig. 1. Schematic diagram of the 3-HP and byproducts biosynthetic pathway used in this

4

study. Glycerol was converted by glycerol dehydratase (comprising the subunits DhaB1,

5

DhaB2 and DhaB3) and glycerol dehydratase reactivase (comprising the subunits GdrA and

6

GdrB) to yield 3-HPA. The 3-HPA was dehydrogenated to 3-HP by aldehyde dehydrogenase

7

(Kgsadh). 1,3-PDO is produced by aldehyde reductase (YqhD) from 3-HPA. Acetate is

8

produced by phosphate acetyltransferase (Pta) and acetate kinase (ackA) from Acetyl-CoA.

9

The catalytic activity of GDHt was controlled by fine-tuning expression of the gene dhaB1

10

(multiple arrows). Two plasmids (pACYC and pUC) were utilized to express GDHt and

11

ALDH.

12 13

Fig. 2. Normalized relative specific enzyme activity for strains DUBGK (A) and HGL_BK1

14

(B). The standard deviations of measurements were derived from three technical replicates.

15

(C and D) Time-course fermentation profile for strains DUBGK (C) and HGL_BK1 (D) in

16

modified glycerol minimal medium for 24 h. The left y-axis and y-offset represent the log

17

scale OD600 and the glycerol concentration (g/L), respectively. The right y-axis represents the

18

metabolite (3-HP, acetate and 1,3-PDO) concentration (g/L). The x-axis represents culture

19

time (h). The error bars indicate standard deviations of measurements from three independent

20

cultures. Circle: OD600; rectangle: glycerol; diamond: 3-HP; up triangle: acetate; down

21

triangle: 1,3-PDO.

22 23

Fig. 3. (A) SDS-PAGE for strains HGL_BK 1–5 expressing various levels of dhaB1. The

24

marked band indicates the expression of DhaB1 (60.67 kDa) in the total fraction of crude

25

lysate. The other strong band indicates the expression of ALDH (50.84 kDa) (B) The


Page 20 of 38

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1

production of 3-HP for strains HGL_BK1–5 after 24 h. The error bars indicate standard

2

deviations of measurements from three independent cultures. (C) Time-course fermentation

3

profile for strains HGL_BK4 in modified glycerol minimal medium for 24 h. The left y-axis

4

and y-offset represent the log scale OD600 and the glycerol concentration (g/L), respectively.

5

The right y-axis represents the metabolite (3-HP, acetate and 1,3-PDO) concentration (g/L).

6

The x-axis represents culture time (h). The error bars indicate standard deviations of

7

measurements from three independent cultures. Circle: OD600; rectangle: glycerol; diamond:

8

3-HP; up triangle: acetate; down triangle: 1,3-PDO. (D) Predicted expression level and

9

normalized relative specific GDHt activity for strains HGL_BK1–5. The standard deviations

10

of measurements were derived from three technical replicates. (E) Normalized amount of

11

dhaB1 transcript for strains HGL_BK1–5. The standard deviations of measurements were

12

derived from three technical replicates. (F) Growth curve for strains HGL_BK1–5 during

13

fermentation for 9 h. The error bars indicate standard deviations of measurements from three

14

independent cultures. Circle: HGL_BK1; rectangle: HGL_BK2; cross: HGL_BK3; diamond:

15

HGL_BK4; up triangle: HGL_BK5.

16 17

Fig. 4. 3-HP (A) and byproduct (B) concentrations (g/L) for strains HGL_BK4 and

18

HGL_DBK4. The 3-HP production of strain HGL_DBK4 increased and byproduct formation

19

decreased compared with strain HGL_BK4. (C) Time-course (48 h) fermentation profile for

20

strain HGL_DBK4 in modified glycerol minimal medium. The left y-axis and y-offset

21

represent the log scale OD600 and the glycerol concentration (g/L), respectively. The right y-

22

axis represents metabolite (3-HP, acetate and 1,3-PDO) concentration (g/L). The x-axis

23

represents the culture time (h). The error bars indicate standard deviations for measurements

24

from three independent cultures. Circle: OD600; rectangle: glycerol; diamond: 3-HP; up

25

triangle: acetate; down triangle: 1,3-PDO.



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

1 2

Fig. 5. Time-course (30 h) fed-batch fermentation profile for strain HGL_DBK4 in modified

3

minimal M9 supplemented with glycerol and glucose. The left y-axis represents the log scale

4

OD600. The right y-axis represents metabolite (3-HP, acetate and 1,3-PDO) concentration

5

(g/L). The x-axis represents culture time (h). The error bars indicate standard deviations for

6

measurements from two independent cultures. Circle: OD600; rectangle: glycerol; diamond: 3-

7

HP; up triangle: acetate; down triangle: 1,3-PDO.

8 9


Page 22 of 38

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1

Tables

2

Table 1. 5′ UTR sequences for expression of 3-HP biosynthesis enzymes.

3 4

Gene

5′′ UTR sequence (5′′-3′′)a

Predicted expression level (a. u.)

dhaB1-V1

CCATAGGTCAAAAGGAGCATCACAA

1,357,410.09

dhaB1-V2

CCATAGGTCAAAAGGAGCATCACCA

570,446.18

dhaB1-V3

ATTTGCTCCAAAAGGAGCATATCGA

26,318.41

dhaB1-V4

CATGCGCTAAAACAGAAGCATCAGTG

1,259.90

dhaB2

AATAAGCAACAAAAAGGAGGAAAAG

5,091,545.46

kgsadh

ATTTTAGGCAAAAGGAGGATCTATC

2,888,200.21

a

Red letters indicate the ribosome binding site.

5



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1

Page 24 of 38

Table 2. Strains and plasmids used in the study. Name Strains Mach1-T1

R

E. coli W DUBGK HGL_BK1 HGL_BK2 HGL_BK3 HGL_BK4 HGL_BK5 HGL_DBK4

Relevant characteristics -

Source -

F φ80(lacZ)∆M15 ∆lacX74 hsdR(rK +

mK ) ∆recA1398 endA1 tonA Acid tolerant strain E. coli W/pDK7(p15A)_DhaB123,gdrAB /pUC_KGSADH E. coli W/pACYC_B1/pUC_K E. coli W/pACYC_B2/pUC_K E. coli W/pACYC_B3/pUC_K E. coli W/pACYC_B4/pUC_K E. coli W/pACYC_B0/pUC_K HGL_BK4/∆ackA-pta/∆yqhD

Plasmids pDK7 (p15A) p15A ori, CmR, Ptac-dhaB123-gdrAB _DhaB123, gdrAB pUC_KGSADH pMB1 ori, KanR, Plac-kgsadh pACYC_Duet Expression vector; p15A ori, CmR pACYC_B0 pACYC_B1

pACYC_B2

pACYC_B3

pACYC_B4 pUC/K

p15A ori, CmR, Ptac-SynUTRdhaB2-dhaB2dhaB3-gdrA-gdrB p15A ori, CmR, Ptac-SynUTR1dhaB1dhaB1- Ptac-SynUTRdhaB2-dhaB2-dhaB3gdrA-gdrB p15A ori, CmR, Ptac-SynUTR2dhaB1dhaB1- Ptac-SynUTRdhaB2-dhaB2-dhaB3gdrA-gdrB p15A ori, CmR, Ptac-SynUTR3dhaB1dhaB1- Ptac-SynUTRdhaB2-dhaB2-dhaB3gdrA-gdrB p15A ori, CmR, Ptac-SynUTR4dhaB1dhaB1- Ptac-SynUTRdhaB2-dhaB2-dhaB3gdrA-gdrB pMB1 ori, KanR, Ptac-SynUTRkgsadhkgsadh

2


Invitrogen KCTC1039 10

This study This study This study This study This study This study

49 50

Novagen This study This study

This study

This study

This study This study

Page 25 of 38

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1


Table 3. Primers used in the study. Name pACYC-F pACYC-B O-dhaB2-F1 O-dhaB2-F2 (speI) O-dhaB2-B (pacI) O-dhaB1-F1 O-dhaB1-F1-V2 O-dhaB1-F1-V3 O-dhaB1-F1-V4 O-dhaB1-B1 O-dhaB1-B2 pUC-F pUC-B O-kgsadh-F1 O-kgsadh-F2 O-kgsadh-B

Sequence (5’-3’)a caggatccgaattcgagctcg ctggttACTAGTaagggagagcgtcgagatcc ggaattgtgagcggataacaattaataagcaacaaaaaggaggaaaaggtgcaacagacaa cccaaattcag cccaacACTAGTttgacaattaatcatcggctcgtataatgtgtggaattgtgagcggata acaatt tctctcTTAATTAAaagctttctagatcagtttctctcacttaacg ggaattgtgagcggataacaattccataggtcaaaaggagcatcacaaatgaaaagatcaaa acgatttgcagtactgg ggaattgtgagcggataacaattccataggtcaaaaggagcatcaccaatgaaaagatcaaa acgatttgcagtactgg ggaattgtgagcggataacaattatttgctccaaaaggagcatatcgaatgaaaagatcaaaac gatttgcagtactgg ggaattgtgagcggataacaattcatgcgctaaaacagaagcatcagtgatgaaaagatcaaa acgatttgcagtactgg ggttcagcccgacaccattgaataacgcaaaaaaccccgcttcggcggggttttttcgc ccaacaACTAGTgcgaaaaaaccccgccgaag ttgccgttcggtcttgccggctacgcgtt acacacacgagCTCGAGtgagctaactcacattaattgcgttgc gtgtggaattgtgagcggataacaattattttaggcaaaaggaggatctatcatggctaacgtg acttatacggatacg gagttagctcactcGAGCTCttgacaattaatcatcggctcgtataatgtgtggaattgtga gcggataacaatt ccggttcgcttgctgtcc

C-yqhD-F C-yqhD-B

agtgcatgatgttaatcataaatgtcggtgtcatcatgcgctacgctctactagtgctggagcga actgc caatccctgcacccagttctacaccctgagacgctgcaacacggtggagtactcgcggttgac tg gtgcaaattcacaactcagcg gcaccacgttgtcttccagc caggcagatcgttctctgccctcatattggcccagcaaagggagcaagtactagtgctggagc gaactgc ctgccgtccagaccgtagtcggagaggtgggtcggcacgcctaattgctcggagtactcgcg gttgactg gtgtacgtgcgaaaggcgtct gctccggtgaggtgttgtg

Q-rrsA-F Q-rrsA-R Q-dhaB1-F

catctgatactggcaagcttgagt gtttgctccccacgctttc ctgagccgcagcggctttgagg

D-ackApta-F D-ackApta-B C-ackApta-F C-ackApta-B D-yqhD-F D-yqhD-B



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Q-dhaB1-R 1 2

a

ttattcaatggtgtcgggctgaaccacgcc

Underlined letters indicate enzyme sites for cloning.

3


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Graphic Abstract 166x83mm (150 x 150 DPI)



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Figure 1 243x154mm (150 x 150 DPI)


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Figure 2 168x51mm (300 x 300 DPI)



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Figure 3 167x106mm (300 x 300 DPI)


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Figure 4 164x64mm (300 x 300 DPI)



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Figure 5 64x56mm (300 x 300 DPI)


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Escherichia coli

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