Metabolic engineering of a homoserine-derived non-natural pathway

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Metabolic engineering of a homoserine-derived non-natural pathway for the de novo production of 1,3-propanediol from glucose Weiqun Zhong, Ye Zhang, Wenjun Wu, Dehua Liu, and ZHEN CHEN ACS Synth. Biol., Just Accepted Manuscript • DOI: 10.1021/acssynbio.9b00003 • Publication Date (Web): 25 Feb 2019 Downloaded from http://pubs.acs.org on February 28, 2019

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ACS Synthetic Biology

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Metabolic engineering of a homoserine-derived non-natural pathway for the de

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novo production of 1,3-propanediol from glucose

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Weiqun Zhong1, Ye Zhang1, Wenjun Wu1, Dehua Liu1,2,3, and Zhen Chen1,2,3*

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1. Key Laboratory of Industrial Biocatalysis (Ministry of Education), Department of

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Chemical Engineering, Tsinghua University, Beijing 100084, China

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2. Tsinghua Innovation Center in Dongguan, Dongguan 523808, China

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3. Center for Synthetic and Systems Biology, Tsinghua University, Beijing 100084,

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China

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*Corresponding author: Zhen Chen

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Tel: +86-10-62772130.

E-mail: [email protected]

Fax: +86-10-62792128

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ACS Paragon Plus Environment

ACS Synthetic Biology 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

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ABSTRACT

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Engineering a homoserine-derived non-natural pathway allows heterologous

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production of 1,3-propanediol (1,3-PDO) from glucose without adding expensive

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vitamin B12. Due to the lack of efficient enzymes to catalyze the deamination of

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homoserine and the decarboxylation of 4-hydroxy-2-ketobutyrate, the previously

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engineered strain can only produce 51.5 mg/L 1, 3-PDO using homoserine and

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glucose as co-substrates. In this study, we systematically screened the enzymes

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from different protein families to catalyze the two corresponding reactions and

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further optimized the selected enzymes by protein engineering. Together with the

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improvement of homoserine supply by systematic metabolic engineering, an

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engineered Escherichia coli strain with an optimal combination of aspartate

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transaminase (aspC) from E. coli, pyruvate decarboxylase (pdc) from Zymomonas

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mobilis, and alcohol dehydrogenase yqhD from E. coli, can produce 0.32 g/L 1,3-

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PDO from glucose in shake flask cultivation. The titer of 1,3-PDO was further

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increased to 0.49 g/L or 0.63 g/L by introducing a point mutation of I472A into pdc

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gene or constructing a fusion protein between aspC and pdc. This study lays the

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basis for developing a potential process for 1,3-PDO production from sugars

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without using expensive coenzyme B12.

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Keywords: 1,3-propanediol, non-natural pathway, homoserine, enzyme screening,

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protein engineering, pathway optimization.

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1,3-Propanediol (1,3-PDO) is a very important chemical which has been widely used

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in different industrial areas, such as solvents, adhesives, antifreeze, and coatings.1,2

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Especially, 1,3-PDO can be used as a monomer for the synthesis of several high-

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performance polymers including the well-known polytrimethylene terephthalate (PTT).

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Biological production of 1,3-PDO from renewable resources has received broad

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attention in the past two decades.3,4 Natural producers can only utilize glycerol as

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substrate to produce 1,3-PDO, mainly by two consecutive enzymatic reactions: (1)

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dehydration of glycerol to 3-hydroxypropionaldehyde (3-HPA) by glycerol dehydratase;

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and (2) reduction of 3-HPA to 1,3-PDO by 1,3-PDO dehydrogenase.2 Dupont and

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Genencor has previously constructed a recombinant Escherichia coli to directly convert

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glucose into 1,3-PDO via the combination of a glycerol synthesis module from

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Saccharomyces cerevisiae and a 1,3-PDO synthesis module from Klebsiella

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pneumoniae.1,5 Although the engineered strain can produce 1,3-PDO with high titer and

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yield, the necessity to add expensive vitamin B12 during fermentation significantly

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increase the whole production cost. Most of the commonly used industrial chassis, such

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as E. coli, S. cerevisiae, and Corynebacterium glutamicum, cannot synthesize

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coenzyme B12, an important cofactor of glycerol dehydratase. Construction of

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heterologous vitamin B12 synthesis pathways in these organisms is highly challenging

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due to the high complexity of B12 synthesis pathways encoded by more than 25 genes.6

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Although a B12-independent glycerol dehydratase was discovered in 2003, this enzyme

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is highly sensitive to oxygen and direct utilization of this enzyme for 1,3-PDO

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production from glucose has not been demonstrated so far.7 3



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A glycerol-independent route to produce 1,3-PDO from sugars was recently proposed

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by our group and two filed patents.8,9,10 This non-natural metabolic pathway converts

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the natural metabolic precursor homoserine into 1,3-PDO via three consecutive

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reactions: (1) deamination of homoserine to 4-hydroxy-2-ketobutyrate; (2)

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decarboxylation of 4-hydroxy-2-ketobutyrate to 3-HPA; (3) reduction of 3-HPA to 1,3-

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PDO (Figure 1). This metabolic pathway has the same theoretical yield as the glycerol-

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dependent pathway but does not require complicated cofactor like coenzyme B12. High

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production of homoserine has been demonstrated in different chassis like E. coli and C.

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glutamicum.11 Thus, this metabolic pathway can be easily introduced into these chassis

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for 1,3-PDO overproduction. However, the main challenge for constructing this non-

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natural pathway is the lack of natural enzymes to efficiently catalyze the deamination

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of homoserine and the decarboxylation of 4-hydroxy-2-ketobutyrate. We have

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previously engineered a glutamate dehydrogenase (GDH) mutant (K92V/T195S) to

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catalyze the oxidative deamination of aspartate, enabling an accumulation of 51.5 mg/L

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1,3-PDO from glucose and homoserine by a recombinant E. coli.8 The titer, however,

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is still too low for practical application.

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In this study, we combine enzyme screening, protein engineering, and combinatorial

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pathway engineering to increase the efficiency of the synthetic pathway to further

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increase the production of 1,3-PDO. Together with the improvement of homoserine

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supply, an engineered E. coli strain with an optimal combination of aspartate

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transaminase (aspC) from E. coli, pyruvate decarboxylase (pdc) from Zymomonas

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mobilis, and alcohol dehydrogenase yqhD from E. coli can produce the highest amount 4


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of 1,3-PDO from glucose in shake flask cultivation. The titer of 1,3-PDO was further

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increased by engineering the specificity of pyruvate decarboxylase and constructing a

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fusion protein between aspartate transaminase and pyruvate decarboxylase to provide a

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potential metabolic channeling for 4-hydroxy-2-ketobutyrate.

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RESULTS AND DISCUSSION

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Selection of enzymes for the decarboxylation of 4-hydroxy-2-ketobutyrate

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The non-natural 1,3-PDO synthesis pathway is comprised of three enzymatic reactions

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(Figure 1). Reduction of 3-HPA to 1,3-PDO can be efficiently catalyzed by the non-

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specific alcohol dehydrogenase (yqhD) from E. coli.8 However, no natural enzymes

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have been previously reported to efficiently catalyze the decarboxylation of 4-hydroxy-

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2-ketobutyrate. In this study, we first screened the activity for 4-hydroxy-2-

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ketobutyrate decarboxylation from α-keto acid decarboxylase superfamily (E.C. 4.1.1-).

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Several decarboxylases shown broad substrate specificity were evaluated, including

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pyruvate decarboxylase from Zymomonas mobilis (pdc),12 α-ketoisovalerate

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decarboxylase from Lactococcus lactis (kivd),13 benzoylformate decarboxylase from

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Pseudomonas putida (mdlC).14 Since no commercial 4-hydroxy-2-ketobutyrate was

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available, we evaluated the efficiency of these decarboxylases based on in vivo 1,3-

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PDO production from homoserine. In our previous study, we have constructed a mutant

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glutamate dehydrogenase (gdhK92V/T195S) which can catalyze the oxidative deamination

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of homoserine to 4-hydroxy-2-ketobutyrate.8 We synthesized all of the selected

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decarboxylases with optimized codons and co-expressed them with gdhK92V/T195S and 5



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yqhD under tac promoter in plasmid pXMJ19 (Figure 2A). All of the constructed

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plasmids were transformed into E. coli strain W02, a quadruple E. coli mutant (ΔldhA

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ΔadhE ΔthrB ΔmetA) blocking the pathways for lactate and ethanol synthesis and

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homoserine consumption. When cultured in M9Y medium with the addition of 5 g/L

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homoserine, all of the constructed recombinants can produce 1,3-PDO (Figure 2B).

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Strain W04 harboring pdc produced the highest amount of 1,3-PDO (~ 0.31 g/L) while

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strain W05 and W06 accumulated < 0.1 g/L 1,3-PDO. Strain W04 also accumulated ~

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5.4 g/L ethanol and a marginal amount of acetate. Contrarily, strain W05 and W06

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accumulated > 5 g/L acetate with < 1.5 g/L ethanol. Since adhE gene has been knocked

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out in strain W02, ethanol production by strain W04, W05, and W06 should be

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attributed to the decarboxylation of pyruvate by the promiscuous activities of the

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introduced α-keto acid decarboxylases. Since pyruvate decarboxylase (pdc) was shown

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to be the most efficient enzyme for decarboxylation of 4-hydroxy-2-ketobutyrate under

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the testing condition, it was selected for further study.

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Selection of enzymes for the deamination of homoserine

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Although glutamate dehydrogenase (gdhK92V/T195S) can catalyze the oxidative

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deamination of homoserine, the catalytic efficiency of this enzyme for homoserine is

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very poor (Km > 1.0 M),8,31 which significantly limits the efficiency of the engineered

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pathway. A more efficient enzyme for the deamination of homoserine is highly

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demanded. In nature, three different enzyme families can transfer amino acid into the

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corresponding α-keto acid, namely: (1) amino acid dehydrogenase; (2) amino acid 6


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transaminase (aminotransferases); (3) amino acid deaminase (oxidase). Thus, different

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proteins from these enzyme families were co-expressed with pdc and yqhD under tac

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promoter in plasmid pXMJ19 (Figure 3A). The constructed plasmids were transformed

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into E. coli strain W02 and the recombinant strains were cultured in M9Y medium with

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the addition of 5 g/L homoserine.

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Amino acid dehydrogenases catalyze the NAD(P)-dependent reversible deamination of

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L-amino acids into α-keto acids. Different from glutamate dehydrogenase that is highly

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specific to glutamate, valine dehydrogenase and leucine dehydrogenase of the ELFV

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dehydrogenase family are able to use a broad scope of substrates.15, 16, 17 We synthesized

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valine dehydrogenase from Streptomyces cinnamonensis (vdh)

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dehydrogenase from Geobacillus stearothermophilus (ldh) 17 and tested their activities

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toward oxidative deamination of homoserine. Both of the purified valine

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dehydrogenase and leucine dehydrogenase can use homoserine as a substrate with Km

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value of 30.8 mM and 44.7 mM and Vmax value of 2.7 μmol/min/mg protein and 1.3

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μmol/min/mg protein at optimum pH 11. However, the activities were significantly

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reduced (~ 0.072 μmol/min/mg protein for VDH and ~ 0.054 μmol/min/mg protein for

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LDH) at pH 7. When cultured in M9Y medium with the addition of 5 g/L homoserine,

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strain W07 harboring ldh and strain W08 harboring vdh accumulated 0.35 g/L and 0.57

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g/L 1,3-PDO, indicating that both ldh and vdh are more efficient than gdhK92V/T195S (0.31

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g/L 1,3-PDO for strain W04 harboring gdhK92V/T195S) (Figure 3B).

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and leucine


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Amino acid transaminases catalyze the reversible transamination between an amino

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acid and an α-keto acid. Several amino acid transaminases with broad substrate

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spectrum were tested in this study, including an aspartate transaminase from E. coli

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(aspC),18 a branched-chain amino acid transaminase from E. coli (ilvE),18 a mutated

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alanine transaminase from E. coli (alaCA142P/Y275D),19 an omega-transaminase from

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Vibrio fluvialis (TA51),20 and a serine-pyruvate aminotransferase gene from Mus

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musculus (Agxt).21 When cultured in M9Y medium with the addition of 5 g/L

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homoserine, strain W09 harboring aspC, strain W10 harboring ivlE, and strain W12

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harboring TA51 produced higher amount of 1,3-PDO than strain W04 harboring

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gdhK92V/T195S (0.56 g/L, 0.39 g/L, and 0.69 g/L vs 0.31 g/L) (Figure 3C).

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Amino acid deaminases/oxidases (LAAO) are flavoenzymes catalyzing the irreversible

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deamination of an amino acid to the corresponding α-keto acids. Three LAAOs with

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broad substrate spectrum were tested in this study, namely L-amino acid oxidase from

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Rhodococcus opacus (RoAo),22 L-amino acid oxidase from Proteus vulgaris (PvAo),23

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L-amino acid oxidase from Proteus mirabilis (PmAo).24 When cultured in M9Y

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medium with the addition of 5 g/L homoserine, strain W14 harboring RoAo, strain W15

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harboring PvAo, and strain W16 harboring PmAo produced 0.17 g/L, 0.26 g/L, and 0.20

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g/L 1,3-PDO (Figure 3D). Expression of catalase from Geobacillus sp. in these strains

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to catalyze the dismutation of H2O2 during amino acid oxidation did not further improve

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1,3-PDO production (data not shown). Thus, LAAOs are not efficient for 1,3-PDO

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production as compared to glutamate dehydrogenase (gdhK92V/T195S).

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Construction of strains for the direct production of 1,3-PDO from glucose

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All of the previously constructed strains produced 1,3-PDO in M9Y medium with the

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additionally supplemented homoserine. To directly produce 1,3-PDO from glucose, we

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overexpressed

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dehydrogenase gene thrAG433R from E. coli and an aspartate-insensitive pyruvate

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carboxylase from C. glutamicum (pycP458S) in plasmid pACYC 184. Plasmid pACYC-

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thrAG433R-pycP458S was transformed into E. coli strain W02, giving strain W03. Strain

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W03 accumulated a significantly higher amount of homoserine than strain W02 (1.34

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g/L vs 0.34 g/L homoserine). Five plasmids allowing high accumulation of 1,3-PDO in

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strain W02 were transformed into strain W03, resulting in strain W17 (pXMJ-

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gdhK92V/T195S-pdc-yqhD), W18 (pXMJ-vdh-pdc-yqhD), W19 (pXMJ-aspC-pdc-yqhD),

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W20 (pXMJ- ilvE-pdc-yqhD), and W21 (pXMJ-TA51-pdc-yqhD).

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The five strains were cultivated in M9Y medium without additionally supplemented

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homoserine. All of the five strains produced lower amounts of 1,3-PDO than the

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corresponding W02-derived strains cultivated in M9Y medium with 5 g/L homoserine

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(Figure 4). This is probably due to that all of the used amino acid dehydrogenases or

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amino acid transaminases showed high Km value for homoserine ( >30 mM or 3.57

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g/L) while all of the W03-derived strains accumulated a relatively low amount of

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homoserine (< 1 g/L). Among all of the tested strains, strain W19 overexpressing aspC,

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pdc and yqhD accumulated the highest amount of 1,3-PDO (0.32 g/L) when cultivated

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threonine-insensitive

bifunctional

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aspartokinase/homoserine


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in M9Y medium without the additionally supplemented homoserine. This strain was

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selected for further optimization.

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Protein engineering of pyruvate decarboxylase to increase 1,3-PDO production

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Although strain W19 can accumulate 0.32 g/L 1,3-PDO using glucose as the sole

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carbon source, it also accumulated a high amount of ethanol (4.9 g/L). High activity of

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pyruvate decarboxylase toward pyruvate decarboxylation would significantly redirect

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metabolic flux toward ethanol production, reducing the yield of homoserine and 1,3-

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PDO. Thus, it is necessary to enhance the specific activity of pyruvate decarboxylase

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toward 4-hydroxy-2-ketobutyrate decarboxylation by protein engineering. Two

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residues, I472 and I476, which located in the substrate’s binding center of pyruvate

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decarboxylase, have been previously identified to be involved in the binding of

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pyruvate (Figure 5A).12 Multiple sequence analysis show that these two residues are

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highly conserved within pyruvate decarboxylases family. In benzoylformate

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decarboxylase, these two residues are mutated to Ala and Phe, allowing the binding of

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large substrates like aromatic α-keto acids.12 Thus, three mutated pyruvate

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decarboxylases, pdcI472A, pdcI476F, and pdcI472A/I476F, were constructed in this study.

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Compared to the wildtype, the activity toward pyruvate decarboxylation was reduced

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by 58.3%, 84.2%, and 98.3% for pdcI472A, pdcI476F and pdcI472A /I476F, respectively.12

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Consequently, when cultured in M9Y medium, ethanol production by strain W22

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harboring pdcI472A, strain W23 harboring pdcI476F, strain W24 harboring pdcI472A /I476F

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was reduced by 46.9%, 55.4%, and 84.9% (Figure 5). 1,3-PDO production and 10


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homoserine accumulation by strain W22 was increased by 52.8% (0.49 g/L vs 0.32 g/L)

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and 32.9% (0.93 g/L vs 0.70 g/L) compared to strain W19. However, 1,3-PDO

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production by strain W23 and strain W24 was significantly reduced compared to strain

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W19, indicating that introduction I476F mutation also significantly reduce the activity

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of pyruvate decarboxylase toward 4-hydroxy-2-ketobutyrate decarboxylation.

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Construction of fusion protein to increase 1,3-PDO production

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To further increase the efficiency of the synthetic pathway, we tried to construct fusion

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proteins between aspartate transaminase and pyruvate decarboxylase. Construction of

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fusion protein is a powerful strategy to enhance pathway efficiency by promoting the

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transfer of intermediates between pathway enzymes.25 Specifically, the fusion of

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aspartate transaminase (aspC) and pyruvate decarboxylase (pdc) may prevent the

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diffusion of 4-hydroxy-2-ketobutyrate, increasing the efficiency of the subsequent

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decarboxylation reaction and reducing the formation of ethanol. A flexible linker

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(GGGGS)3 was inserted between the C-terminal of aspC and the N-terminal of pdc or

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pdcI472A, giving strain W25 (aspC-linker-pdc) and W26 (aspC-linker-pdcI472A). When

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cultivated in M9Y medium, strain W25 produced a 1.97-fold higher amount of 1,3-

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PDO than strain W19 (0.63g/L vs 0.32 g/L), indicating that fusion of aspartate

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transaminase and pyruvate decarboxylase could significantly promote 1,3-PDO

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production (Figure 6). However, the fusion of aspartate transaminase and the mutated

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pyruvate decarboxylase (I472A) did not further promote 1,3-PDO production probably

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due to the reduced solubility of the fusion protein which resulted in the lower activity 11



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of aspC and the higher accumulation of homoserine (Figure 6). Changing the length of

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the linker did not further increase the production of 1,3-PDO.

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CONCLUSION

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In this study, a homoserine-derived non-natural pathway for 1,3-PDO production was

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optimized by combining enzyme screening, protein engineering, and pathway

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engineering. Especially, aspartate transaminase (aspC) from E. coli and pyruvate

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decarboxylase (pdc) from Z. mobilis were selected as the most promising combination

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for the engineered artificial pathway. Aspartate transaminase and several other selected

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enzymes in this study showed improved kinetics for homoserine deamination compared

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to the previously engineered glutamate dehydrogenase (Km of homoserine ~ 72 mM

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for aspC vs > 1.0 M for gdhK92V/T195S).12,31 However, all of these natural enzymes

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including the wildtype pyruvate decarboxylase show broad substrate spectrum and

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prefer to use their native substrates, resulting in the accumulation of large amounts of

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byproducts. Reduce the activity of pyruvate decarboxylase towards pyruvate

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decarboxylation in this study was shown to be efficient to reduce the accumulation of

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ethanol and to partially increase the production of 1,3-PDO. Fusion of aspartate

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transaminase and pyruvate decarboxylase to provide metabolic channeling of 4-

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hydroxy-2-ketobutyrate also significantly increased the production of 1,3-PDO. The

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best engineered E. coli strain can produce 0.63 g/L 1,3-PDO from glucose without

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adding vitamin B12, which is about 10-fold higher than the previously engineered

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strain.8 The present study lays the basis for developing an efficient biological process 12


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for 1,3-PDO production from sugars. Further optimization of aspC and pdc by protein

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engineering to increase the specificity of the enzymes toward homoserine

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transamination and 4-hydroxy-2-ketobutyrate decarboxylation is necessary for

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increasing 1,3-PDO production and reducing the accumulation of other byproducts.26

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Increasing the precursor (homoserine) availability can also be used to promote the

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accumulation of 1.3-PDO. Similar pathways and metabolic engineering strategies can

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be implemented for the production of 3-hydroxypropionic acid by changing alcohol

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dehydrogenase to aldehyde dehydrogenase or ethylene glycol by changing the

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precursor from homoserine to serine.27,14

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METHODS

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Strains and plasmids

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Strains used in this study are listed in Table 1 and plasmids used are listed in

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Supplemental Table 1. All strains used in this study were derived from E. coli K-12

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strain W3110. The in-frame deletions of ldhA, adhE, thrB, and metA genes were

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obtained by CRISPR–Cas9 mediated genome editing as described by Li et al.28

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Construction of artificial 1,3-PDO synthesis pathways were based on an expression

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vector pXMJ19.27 Low-copy plasmid pACYC184 was used as backbone for

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overexpressing mutated thrA gene (G433R) from E. coli and pyc gene (P458S) from

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Corynebacterium glutamicum. Gibson assembly cloning kit (NEB) was used to

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construct all of the plasmids.29

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Plasmid construction 13



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To construct plasmid pACYC-thrAG433R-pycP458S, the mutated thrA gene (G433R) with

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its native promoter was amplified from a classical threonine hyperproducer E. coli

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ATCC 21277 and the mutated pyc gene (P458S) was amplified from C. glutamicum

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AC 256. The two fragments were inserted into the NcoI/EcoRI sites of pACYC184 by

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the standard protocol of Gibson assembly.

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Plasmid pXMJ-gdhK92V/T195S-pdc-yqhD was constructed by PCR-amplifying a DNA

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fragment containing gdhAK92V/T195S-pdc-yqhD operon from pZA-gdhAK92V/T195S-pdc-

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yqhD.8 The DNA fragment was inserted into the XbaI/EcoRI sites of pXMJ19 by

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Gibson assembly. Plasmids pXMJ-gdhK92V/T195S-kivd-yqhD and pXMJ-gdhK92V/T195S-

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mdlC-yqhD were obtained by replacing the pdc gene in pXMJ-gdhK92V/T195S-pdc-yqhD

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by the codon-optimized kivd gene from Lactococcus lactis and the mdlC gene from

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Pseudomonas putida by Gibson assembly. The codon-optimized kivd gene and mdlC

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gene were amplified from plasmids pEC-P1-LlKivD-yqhD and pEC-P1-PpMdlc-

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yqhD.14

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Plasmids pXMJ-vdh-pdc-yqhD and pXMJ-ldh-pdc-yqhD were obtained by replacing

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the gdhK92V/T195S gene in pXMJ-gdhK92V/T195S-pdc-yqhD by the codon-optimized vdh

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gene from Streptomyces cinnamonensis and the ldh gene from Geobacillus

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stearothermophilus by Gibson assembly. The codon-optimized vdh gene and ldh gene

288

were amplified from plasmids pEC-P1-ScVdh-PpMdlc-yqhD and pEC-P1-GsLdh-

289

PpMdlc-yqhD.14 Plasmids pXMJ-aspC-pdc-yqhD, pXMJ-ilvE-pdc-yqhD, pXMJ-

290

alaCA142P/Y275D-pdc-yqhD, pXMJ-TA51-pdc-yqhD, and pXMJ-Agxt-pdc-yqhD were 14


Page 14 of 29

Page 15 of 29 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


291

obtained by replacing the gdhK92V/T195S gene in pXMJ-gdhK92V/T195S-pdc-yqhD by the

292

aspC gene and ilvE gene from E. coli, the mutated alaC gene (A142P/Y275D) from E.

293

coli, the codon-optimized omega-transaminase gene from Vibrio fluvialis (TA51,

294

Uniprot No. F2XBU9) and the serine-pyruvate aminotransferase gene from Mus

295

musculus (Agxt, Uniprot No. O35423) by Gibson assembly. AspC gene and ilvE gene

296

were amplified from E. coli W3110. The mutated alaC gene (A142P/Y275D)

297

(Supplemental Sequence 1), codon-optimized TA51 gene (Supplemental Sequence 2)

298

and Agxt gene (Supplemental Sequence 3) were synthesized by Qinglan Biotech (WuXi)

299

co., Ltd.

300

Plasmids pXMJ-RoAo-pdc-yqhD, pXMJ-PvAo-pdc-yqhD, and pXMJ-PmAo-pdc-

301

yqhD were obtained by replacing the gdhK92V/T195S gene in pXMJ-gdhK92V/T195S-pdc-

302

yqhD by the codon-optimized L-amino oxidase gene from Rhodococcus opacus (RoAo,

303

Uniprot No. Q8VPD4), L-amino oxidase gene from Proteus vulgaris (PvAo, Uniprot

304

No. Q9LCB2), L-amino oxidase gene from Proteus mirabilis (PmAo, Uniprot No.

305

B2ZHY0) by Gibson assembly. The codon-optimized RoAo (Supplemental Sequence

306

4), PvAo (Supplemental Sequence 5), and PmAo (Supplemental Sequence 6) genes

307

were synthesized by Qinglan Biotech (WuXi) co., Ltd.

308

Plasmids pXMJ-aspC-pdcI472A-yqhD, pXMJ-aspC-pdcI476F-yqhD, and pXMJ-aspC-

309

pdcI472A/I476F-yqhD were obtained by site-directed mutagenesis based on pXMJ-aspC-

310

pdc-yqhD using Gibson assembly kit (NEB). Plasmids pXMJ-aspC-linker-pdc-yqhD

311

and pXMJ-aspC-linker-pdcI472A-yqhD were obtained with the deletion of the stop codon 15



aspC

gene

and

insertion

of

the

Page 16 of 29

linker

sequence

(5

′

312

of

-

313

GGTGGTGGTGGTAGTGGTGGCGGTGGTTCAGGCGGTGGTGGTTCC-3

314

between aspC gene and pdc gene by site-directed mutagenesis using pXMJ-aspC-pdc-

315

yqhD and pXMJ-aspC-pdcI472A-yqhD as template. To construct plasmids for protein

316

overexpression and characterization in E. coli BL21 (DE3), all gene fragments were

317

cloned into pET-28a between restriction sites EcoRI and SalI in frame with the N-

318

terminal his-tag. The detailed method used to construct plasmids is described in the

319

Supporting Information.

320

Table 2.

321

Protein expression and enzyme assay

322

E. coli BL21 (DE3) harboring pET-28a derived plasmids were grown in LB medium

323

with 50 μg/ml Kanamycin at 37 ℃ until OD600 reached 0.6 and gene expression was

324

induced at 20 ℃ for an additional 12–14 h by adding 0.1 mM isopropyl β-D-

325

thiogalactopyranoside (IPTG). Protein purification was conducted by using Ni2+-NTA

326

column (GE Healthcare Bio-Sciences, Piscat-away, NJ) following the manufacturer's

327

instructions. The purity of the enzymes was checked by SDS-PAGE and protein

328

concentrations were quantified via the Bradford assay. The purified enzymes were then

329

used for enzyme characterization.

330

The activities of amino acid dehydrogenases were assayed by the method described by

331

Wu et al. (2018).15 The activities of amino acid transaminases were assayed by the

′ )

The primers used in this study are listed in Supplemental

16


Page 17 of 29 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


332

method described by Walther et al.(2018).18 The activities of pyruvate decarboxylase

333

and its mutants were assayed by the method described by Siegert et al. (2005).12

334

Culture condition and analytical method

335

The modified M9Y medium was used for 1,3-PDO production by E. coli W03-derived

336

strains, consisting of (per liter): 20 g glucose, 12.8 g Na2HPO4·7H2O, 0.5 g NaCl, 3 g

337

KH2PO4, 1 g NH4Cl, 0.25 g MgSO4·7H2O, 14.7 mg CaCl2·2H2O, 2.78 mg FeSO4·7H2O,

338

10 mg thiamine hydrochloride, 0.5 g threonine, 0.5 g methionine, and 5 g yeast extract.

339

M9Y medium supplemented with 5 g/L homoserine was used for 1,3-PDO production

340

by E. coli W02-derived strains. When needed, the medium was supplemented with 50

341

μg/ml chloramphenicol and/or 10 μg/ml tetracycline. Shake-flask fermentation was

342

performed in 500 ml flasks containing 50 ml M9Y minimal medium. Main cultures

343

were inoculated at an initial OD600 of 0.2 from overnight pre-cultures in LB medium.

344

The cell was grown at 30°C and 200 rpm agitation. Expression of 1,3-PDO synthesis

345

pathway was induced by the addition of 1 mM isopropyl β-D-1-thiogalactopyranoside

346

(IPTG) when OD600 reached approximately 0.6.

347

Quantification of glucose, 1,3-PDO and other organic acids were carried out by using

348

High performance liquid chromatography (HPLC) equipped with a Aminex HPX-87H

349

Column (300×7.8 mm) using 5 mM H2SO4 as the mobile phase with a flow rate of 0.6

350

mL/min, and detection via refractive index or UV absorption at 210 nm.30 The

351

extracellular amino acids were quantified by HPLC after derivatizing with 6-

17



carbamate.30

Page 18 of 29

352

Aminoquinolyl-N-Hydroxysuccinimidyl

353

determined at an optical density of 600 nm.

354

ASSOCIATED CONTENTS

355

Supporting information

356

Detailed methods of plasmids construction, plasmids used in this study, custom DNA

357

oligonucleotide primers, and sequences of the synthetic genes.

358

AUTHOR INFORMATION

359

Corresponding author

360

*Tel: +86-10-62772130; E-mail: [email protected]

361

Author contributions

362

Z.C., W.Z., and D.L. proposed the idea and designed the experiments. W.Z., Y.Z., and

363

W.W. performed the experiments. Z.C. and W.Z. wrote the paper. All authors read and

364

approved the final manuscript.

365

Notes

366

The authors declare no competing financial interest.

367

ACKNOWLEDGMENTS

368

This work was supported by the National Natural Science Foundation of China (Grant

369

Nos. 21878172 and 21676156), the Suzhou-Tsinghua Innovation leading Project (Grant 18


Cell

concentration

was

Page 19 of 29 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


370

No. 20171470060), the DongGuan Innovative Research Team Program （ No.

371

201536000100033 ） , and the Tsinghua University’s Initiative Scientific Research

372

Program (Grant No. 20151080362).

373

REFERENCES

374

(1) Nakamura, C.E., and Whited, G.M., (2003) Metabolic engineering for the microbial production of 1,3-propanediol. Curr. Opin. Biotechnol. 14, 454–459.

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(2) Zhang, Y., Chen, Z., and Liu, D. (2017) Production of C2–C4 diols from renewable bioresources: new metabolic pathways and metabolic engineering strategies. Biotechnol. Biofuels 10, 299. (3) Saxena, R.K., Anand, P., Saran, S., and Isar, J. (2009) Microbial production of 1,3propanediol: recent developments and emerging opportunities. Biotechnol. Adv. 27, 895–913. (4) Xin, B., Tao, F., Wang, Y., Liu, H., M,a C., and Xu, P. (2017) Coordination of metabolic pathways: enhanced carbon conservation in 1,3-propanediol production by coupling with optically pure lactate biosynthesis. Metab. Eng. 41, 102–114. (5) Antoniewicz, M.R., Kraynie, D.F., Laffend, L.A., González-Lergier, J., Kelleher, J.K., and Stephanopoulos, G. (2007) Metabolic flux analysis in a nonstationary system: fed-batch fermentation of a high yielding strain of E. coli producing 1,3propanediol. Metab. Eng. 9, 277–292. (6) Fang, H., Li, D., Kang, J., Jiang, P., Sun, J., and Zhang, D. (2018) Metabolic engineering of Escherichia coli for de novo biosynthesis of vitamin B12. Nat. Commun. 9, 4917. (7) Raynaud, C., Sarçabal, P., Meynial-Salles, I., Croux, C., and Soucaille, P. (2003) Molecular characterization of the 1,3-propanediol (1,3-PD) operon of Clostridium butyricum. Proc. Natl. Acad. Sci. USA. 100, 5010-5015.

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(8) Chen, Z., Geng, F., and Zeng, A.P. (2015) Protein design and engineering of a de novo pathway for microbial production of 1,3-propanediol from glucose. Biotechnol. J. 10, 284-289.

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(9) Boisart, C. (2013) Method for the preparation of 1,3-propanediol. EP2540834 A1.

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(10) Xu, J., Saunders, C.W., Green, P.R., and Velasquez, J.E. (2013) Microorganisms and methods for producing acrylate and other products from homoserine. WO/2013/052717 A2. (11) Li, H., Wang, B.S., Zhu, L.H., Cheng, S., Li, Y.R., Zhang, L., Ding, Z.Y., Gu, Z.H., and Shi, G.Y. (2016) Metabolic engineering of Escherichia coli W3110 for L-homoserine production. Process Biochem. 51, 1973-1983. (12) Siegert, P., McLeish, M.J., Baumann, M., Iding, H., Kneen, M.M., Kenyon, G.L., and Pohl, M.(2005) Exchanging the substrate specificities of pyruvate decarboxylase from Zymomonas mobilis and benzoylformate decarboxylase from Pseudomonas putida. Protein Eng. Des. Sel. 18, 345-357. (13) De la Plaza, M., Fernández de Palencia, P., Peláez, C., and Requena, T. (2004) Biochemical and molecular characterization of alpha-ketoisovalerate decarboxylase, an enzyme involved in the formation of aldehydes from amino acids by Lactococcus lactis. FEMS Microbiol. Lett. 238, 367–374. (14) Chen, Z., Huang, J., Wu, Y., and Liu, D. (2016) Metabolic engineering of Corynebacterium glutamicum for the de novo production of ethylene glycol from glucose. Metab. Eng. 33, 12–18. (15) Wu, W., Zhang, Y., Huang, J., Wu, Y., Liu, D., and Chen, Z. (2018) Discovery of a potentially new subfamily of ELFV dehydrogenases effective for L-arginine deamination by enzyme mining. Biotechnol. J. 13, 1700305. (16) Turnbull, A.P., Baker, P.J., and Rice, D.W. (1997) Analysis of the quaternary structure, substrate specificity, and catalytic mechanism of valine dehydrogenase. J. Biol. Chem. 272, 25105–25111. (17) Kataoka, K. and Tanizawa, K. (2003) Alteration of substrate specificity of leucine dehydrogenase by site-directed mutagenesis. J. Mol. Catal. B Enzym. 23, 299–309. (18) Walther T, Calvayrac F, Malbert Y, Alkim C, Dressaire C, Cordier H, and François JM. (2018) Construction of a synthetic metabolic pathway for the production of 2,4-dihydroxybutyric acid from homoserine. Metab. Eng. 45, 237-245. (19) Bouzon, M., Perret, A., Loreau, O., Delmas, V., Perchat, N., Weissenbach, J., Taran, F., and Marlière, P. (2017) A synthetic alternative to canonical one-carbon metabolism. ACS Synth. Biol. 6, 1520-1533. (20) Hernandez, K., Bujons, J., Joglar, J., Charnock, S.J., de María, P.D., Fessner, W.D., and Clapés P. (2017) Combining aldolases and transaminases for the synthesis of 2-amino-4-hydroxybutanoic acid. ACS Catal. 7, 1707-1711. 20


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(21) Li, X.M., Salido, E.C., Shapiro, LJ. (1999) The mouse alanine:glyoxylate aminotransferase gene (Agxt1): cloning, expression, and mapping to chromosome 1. Somat. Cell Mol. Genet. 25, 67-77. (22) Geueke, B., and Hummel, W. (2002) A new bacterial L-amino acid oxidase with a broad substrate specificity: purification and characterization. Enzyme Microb. Technol. 31, 77-87. (23) Takahashi, E., Ito, K., and Yoshimoto, T. (1999) Cloning of L-amino acid deaminase gene from Proteus vulgaris. Biosci. Biotechnol. Biochem. 63, 22442247. (24) Baek, J.O., Seo, J.W., Kwon, O., Seong, S.I., Kim, I.H., and Kim, C.H. (2011) Expression and characterization of a second L-amino acid deaminase isolated from Proteus mirabilis in Escherichia coli. J. Basic Microbiol. 51, 129-135. (25) Fujiwara, R., Noda, S., Tanaka, T., and Kondo, A. (2018) Muconic acid production using gene-level fusion proteins in Escherichia coli. ACS Synth. Biol. 7, 26982705. (26) Chen, Z., and Zeng, A.-P. (2016). Protein engineering approaches to chemical biotechnology. Curr. Opin. Biotechnol. 42, 198–205. (27) Chen, Z., Huang, J., Wu, Y., Wu, W., Zhang, Y., and Liu, D. (2017) Metabolic engineering of Corynebacterium glutamicum for the production of 3hydroxypropionic acid from glucose and xylose. Metab. Eng. 39, 151–158. (28) Li, Y., Lin, Z., Huang, C., Zhang, Y., Wang, Z., Tang, Y.J., Chen, T., and Zhao, X. (2015) Metabolic engineering of Escherichia coli using CRISPR-Cas9 meditated genome editing. Metab. Eng. 31, 13-21. (29) Gibson, D.G., Young, L., Chuang, R.-Y., Venter, J.C., Hutchison, C. a, and Smith, H.O. (2009) Enzymatic assembly of DNA molecules up to several hundred kilobases. Nat. Methods 6, 343–345. (30) Chen, Z., Bommareddy, R.R., Frank, D., Rappert, S., and Zeng, A.-P. (2014) Deregulation of feedback inhibition of phosphoenolpyruvate carboxylase for improved lysine production in Corynebacterium glutamicum. Appl. Environ. Microbiol. 80, 1388–1393. (31) Chen, Z., Geng, F., and Zeng, A.P. (2016) Erratum: Protein design and engineering of a de novo pathway for microbial production of 1,3-propanediol from glucose. Biotechnol. J. 11, 1368-1368.

466 21



467

Page 22 of 29

Table 1. Strains used for this study Strain or plasmid

Description

Reference

Strains W3110

Wildtype

W02

W3110 ΔldhA ΔadhE ΔthrB ΔmetA

W03

ATCC 27325 This study

W02 harboring

pACYC-thrAG433R-pycP458S

This study

W02 harboring

pXMJ-gdhK92V/T195S-pdc-yqhD

This study

W05

W02 harboring

pXMJ-gdhK92V/T195S-kivd-yqhD

This study

W06

W02 harboring pXMJ-gdhK92V/T195S-mdlc-yqhD

This study

W07

W02 harboring pXMJ-ldh-pdc-yqhD

This study

W08

W02 harboring pXMJ-vdh-pdc-yqhD

This study

W09

W02 harboring pXMJ-aspC-pdc-yqhD

This study

W10

W02 harboring pXMJ-ilvE-pdc-yqhD

This study

W04

pXMJ-alaCA142P/Y275D-pdc-yqhD

W11

W02 harboring

W12

W02 harboring pXMJ-TA51-pdc-yqhD

This study

W13

W02 harboring pXMJ-Agxt-pdc-yqhD

This study

W14

W02 harboring pXMJ-RoAo-pdc-yqhD

This study

W15

W02 harboring pXMJ-PvAo-pdc-yqhD

This study

W16

W02 harboring pXMJ-PmAo-pdc-yqhD

This study

W17

W03 harboring pXMJ-

W18

W03 harboring pXMJ-vdh-pdc-yqhD

This study

W19

W03 harboring pXMJ-aspC-pdc-yqhD

This study

W20

W03 harboring pXMJ-ilvE-pdc-yqhD

This study

W21

W03 harboring pXMJ-TA51-pdc-yqhD

This study

W22

W03 harboring pXMJ-aspC-pdcI472A-yqhD

This study

W23

W03 harboring pXMJ-aspC-pdcI476F-yqhD

This study

W24

W03 harboring pXMJ-aspC-pdcI472A/I476F-yqhD

This study

W25

W03 harboring pXMJ-aspC-linker-pdc-yqhD

This study

W26

W03 harboring

gdhK92V/T195S-pdc-yqhD

This study

pXMJ-aspC-linker-pdcI472A-yqhD

468

469

470

471

472 22


This study

This study

Page 23 of 29 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

473


FIGURES

474 475 476 477 478 479 480 481 482 483 484 485 486

Figure 1. Synthetic metabolic pathway for the production of 1, 3-propanediol from homoserine in E. coli. A synthetic module was constructed for the conversion of homoserine to 1, 3-propanediol: (1) deamination of homoserine to 4-hydroxy-2ketobutyrate by amino acid dehydrogenase/transaminase/oxidase; (2) decarboxylation of 4-hydroxy-2-ketobutyrate to 3-hydroxypropionaldehyde by αketo acid decarboxylase; (3) reduction of 3-hydroxypropionaldehyde to 1,3propanediol by alcohol dehydrogenase (yqhD). A feedback-insensitive pyruvate carboxylase from C. glutamicum (pycP458S) and a bifunctional aspartokinase/homoserine dehydrogenase (thrAG433R) from E. coli were overexpressed to increase the availability of homoserine for the synthetic pathway. The red X indicates deletion of the ldhA, adhE, thrB, and metA genes to reduce byproduct formation. 23



487 488

Figure 2. Screening of α-keto acid decarboxylases for 1,3-PDO production. (A)

489

Composition of pathway enzymes constructed in plasmid pXMJ19; (B) 1,3-PDO

490

production; (C) Ethanol accumulation; (D) Acetate accumulation. The cells were

491

cultured in shake flasks with M9Y minimal medium with the addition of 5 g/L

492

homoserine. Data were taken from 72 h of cultivation.

493

24


Page 24 of 29

Page 25 of 29 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


494 495

Figure 3. Screening of enzymes for the deamination of homoserine for 1,3-PDO

496

production. (A) Composition of pathway enzymes constructed in plasmid pXMJ19;

497

(B) 1,3-PDO production with different amino acid dehydrogenases; (C) 1,3-PDO

498

production with different amino acid transaminases; (D) 1,3-PDO production with

499

different amino acid oxidases. The cells were cultured in shake flasks with M9Y

500

minimal medium with the addition of 5 g/L homoserine. Data were taken from 72

501

h of cultivation.

502 503 504 505 506 25



507 508 509 510 511 512

Figure 4. Fermentation profiles of selected strains. (A) Composition of pathway enzymes constructed in plasmid pXMJ19 and plasmid pACYC184; (B) 1,3-PDO production; (C) L-homoserine accumulation; (D) Ethanol accumulation; (E) Acetate accumulation. The cells were cultured in shake flasks with M9Y minimal medium. Data were taken from 72 h of cultivation.

513

26


Page 26 of 29

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514 515 516 517 518 519

Figure 5. Fermentation profiles of mutants with point mutation of pyruvate decarboxylase. (A) The active site of pyruvate decarboxylase (PDB 5TMA); (B) 1,3-PDO production; (C) L-homoserine accumulation; (D) Ethanol accumulation; (E) Acetate accumulation. The cells were cultured in shake flasks with M9Y minimal medium. Data were taken from 72 h of cultivation.

520 521

27



522 523 524 525 526 527 528

Figure 6. Fermentation profiles of mutants with linker between aspartate transaminase and pyruvate decarboxylase. (A) Composition of pathway enzymes constructed in plasmid pXMJ19 and plasmid pACYC184; (B) 1,3-PDO production; (C) L-homoserine accumulation; (D) Ethanol accumulation; (E) Acetate accumulation. The cells were cultured in shake flasks with M9Y minimal medium. Data were taken from 72 h of cultivation.

529 530 531 532 533 534 535 28


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536


For Table of Contents Use Only:

537

538 539

Metabolic engineering of a homoserine-derived non-natural pathway for the de

540

novo production of 1,3-propanediol from glucose

541

Weiqun Zhong1, Ye Zhang1, Wenjun Wu1, Dehua Liu1,2,3, and Zhen Chen1,2,3*

542 543 544

29


Metabolic engineering of a homoserine-derived non-natural pathway

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