Development of a Plasmid-Free Biosynthetic Pathway for Enhanced

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Development of a plasmid-free biosynthetic pathway for enhanced muconic acid production in Pseudomonas chlororaphis HT66 Songwei Wang, Muhammad Bilal, Yuanna Zong, Hong-Bo Hu, Wei Wang, and Xue-Hong Zhang ACS Synth. Biol., Just Accepted Manuscript • DOI: 10.1021/acssynbio.8b00047 • Publication Date (Web): 02 Apr 2018 Downloaded from http://pubs.acs.org on April 3, 2018

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ACS Synthetic Biology

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Development of a plasmid-free biosynthetic pathway for

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enhanced muconic acid production in Pseudomonas chlororaphis

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HT66

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Songwei Wang1, Muhammad Bilal1, Yuanna Zong1, Hongbo Hu1,2, Wei Wang1, Xuehong

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Zhang1*

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1

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Shanghai Jiao Tong University, Shanghai, 200240, China

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2

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Tong University, Shanghai, 200240, China

State Key Laboratory of Microbial Metabolism, School of Life Sciences and Biotechnology,

National Experimental Teaching Center for Life Sciences and Biotechnology, Shanghai Jiao

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*Corresponding author: [email protected] (Xuehong Zhang); Tel: +86-21-3420-6742

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Fax: +86-21-3420-6791

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S Supporting Information ○

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Abstract

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Muconic acid is a platform chemical and an important intermediate in the degradation

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process of a series of aromatic compounds. Herein, a plasmid-free synthetic pathway in

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Pseudomonas chlororaphis HT66 is constructed for the enhanced biosynthesis of muconic

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acid by connecting endogenous ubiquinone biosynthesis pathway with protocatechuate

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degradation pathway using chromosomal integration. Instead of being plasmid and inducer

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dependent, the engineered strains could steadily produce the high muconic acid using

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glycerol as a carbon source. The engineered strain HT-MA6 achieved a 3376 mg/L muconic

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acid production with a yield of 187.56 mg/g glycerol via the following strategies: (1) block

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muconic acid conversion and enhance muconic acid efflux pumping with phenazine

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biosynthesis cluster; (2) increase the muconic acid precursors supply through overexpressing 1

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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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the rate-limiting step and (3) co-express “3-dehydroshikimate-derived” route in parallel with

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“4-hydroxybenzoic acid-derived route” to create a synthetic “metabolic funnel”. Finally,

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based on glycerol feeding strategies, the muconic acid yield reached 0.122 mol/mol glycerol.

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In conclusion, the results suggest that the construction of synthetic pathway with a

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plasmid-free strategy in P. chlororaphis displays a high biotechnological perspective.

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Keywords: Muconic acid; Pseudomonas chlororaphis; biosynthesis; plasmid-free strategy

30 31

Microbial-based metabolic engineering is a powerful and eco-friendly approach for the

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production of high-value compounds from sustainable carbon sources. A series of commercial

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biological products have been successfully developed through the design and construction of

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artificial synthesis pathways in conventional microorganisms, such as Escherichia coli,

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Bacillus, and yeast 1-3. Some Pseudomonas strains could be used as important microbial cell

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factories for the production of valuable compounds

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out for the production of monostearic acid, 2-butyric acid, and 2-quinoxaline formic acid in

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Pseudomonas aeruginosa and P. putida

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rhizosphere 6, 10 is recognized as a safe plant growth promoting rhizobacteria (PGPR) through

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comparative genomics analysis 11. Its wild-type strain is reported to be the highest producer

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of phenazine compounds with simple nutrition and wide-environmental adaptability.

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Moreover, transcriptome and proteome sequencing makes the P. chlororaphis as a choice of

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chassis cell for genetic manipulation and regulation 12.

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P. chlororaphis produces phenazines via shikimate pathway, which serves as a primary

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source of building blocks to produce natural products by microbes. A wide variety of

4-7

. Previously, studies have been carried

8, 9

. P. chlororaphis HT66 isolated from rice

2


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aromatic compounds and phenazine derivatives can be produced from the common precursor

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chorismate via shikimate pathway

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bacteria, plants and fungi serving as a vital intermediate for biosynthesis of aromatic amino

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acid, ubiquinone, formate, and complex natural products (such as polyketides, polyenes or

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terpenoids)

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derivatives was designed in E. coli 18. Despite more efforts have been employed in E. coli, the

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huge demand for glucose sources is not cost-effective, and plasmid-based expression is not

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favorable in fermentation industry.

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P. chlororaphis has been widely engineered as phenazine-producing platform organism due

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to well-characterized physiology and genetics, and fast cell-growth rate using glycerol as a

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carbon source. Glycerol is a major by-product of biodiesel manufacturing process and

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regarded as a promising feedstock for industrial applications. It has a higher degree of

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reduction than glucose and produces twice as much reducing equivalents when converted to

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phosphoenolpyruvate 19. With a deep understanding of the mechanism for the production of

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phenazine compounds and the metabolic regulation of P. chlororaphis, phenazine

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biosynthesis pathway could be modified or metabolized to produce other aromatic

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compounds.

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Muconic acid (MA), a promising precursor to adipic acid, is a platform chemical which is

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used for the synthesis of various plastics and polymers

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intermediate in the degradation process of a series of aromatic compounds via a β-ketoadipate

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pathway. The β-ketoadipate pathway for aromatic compounds degradation has been studied

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extensively in Acinetobacter sp. ADP1

13-15

. Chorismate is a central branch point metabolite in

16, 17

. A synthetic metabolic pathway suitable for the production of chorismate

23

20-22

. MA is a naturally occurring

and P. putida KT2440 3


24-26

. This pathway is only


68

found in soil microorganisms, especially in Pseudomonas species 24, 27. In recent years, many

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improvements in MA production have been made in developing microbial cell factories by

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the construction of non-natural biosynthetic pathways and optimizations of metabolic

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networks 28-31.

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In this study, we utilized the high-efficient shikimate pathway of P. chlororaphis HT66 for

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the biosynthesis of MA by (i) Screening the gene locus candidates for optimized expression

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of genes; (ii) Creation of a high-yielding pathway for 4-HBA production; (iii) Combining the

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ubiquinone biosynthesis and protocatechuate degradation pathway to biosynthesize MA from

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glycerol, using the MA biosynthesis pathway to substitute the phenazine synthesis pathway.

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Next, the “DHS-derived” route was introduced into MA pathway to create a synthetic

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“metabolic funnel” (Figure 1). Finally, different optimizing strategies based on the regulation

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of phenazine biosynthesis were used for MA biosynthesis from glycerol.

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Results and discussion

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Characterization of the β-ketoadipate pathway in P. chlororaphis HT66

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Soli bacteria such as Pseudomonas species and closely related organisms show an intricate

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network of catabolic pathways for aromatic compounds and have been studied extensively 32.

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P. chlororaphis HT66 isolated from rice rhizosphere is able to utilize a range of aromatic

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compounds, such as 3-hydroxybenzoic acid (3-HBA), 4-HBA, protocatechuic acid

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(3,4-HBA), catechol (MC) and gallic acid as sole carbon and energy sources (Figure S1).

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Generally, aromatic compounds are prepared for enzymatic ring cleavage by peripheral

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degradation pathways leading to benzoic acid derivatives, which are subsequently converted

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into tricarboxylic acid cycle intermediates using the β-ketoadipate pathway or the gentisate 4


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pathway 24. P. chlororaphis HT66 can use 4-HBA as a sole carbon source to grow, however,

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this growth ability was found to disappear with the deletion of pobA gene encoding

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4-hydroxybenzoate 3-monooxygenase (Figure 2A). It was confirmed that 4-HBA can be

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converted to protocatechuic acid (PCA) by PobA as shown in Figure 2D. Figure 2D portrays

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a time-course chromatogram of the reaction mixture for crude enzymatic assays. The

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candidate peak of PCA (found at ~15 min) increased as the peak of 4-HBA (found at ~21 min)

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decreased. To further substantiate the PCA formation, the reaction mixture was analyzed

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using UPLC-Q/TOF MS mass spectrometer. The peak of PCA was observed around m/z of

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153.01 (negative ions) indicating that PCA was successfully converted from 4-HBA using

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endogenous pobA. The specific activities and kinetic parameters of purified PobA were

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determined using 2 mM 4-HBA as a substrate. Table 1 shows that the specific activity of

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PobA was 17.25 ± 1.5 U/mg protein, slightly higher than that reported in P. aeruginosa

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(16.85 ± 1.39 U/mg protein). The enzyme assays revealed that PobA (P. chlororaphis) has a

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kcat value of 15.24 ± 1.25 s-1 towards 4-HBA with Km=35.21 ± 4.12 µM, which shows similar

104

substrate affinity with PobA of P. aeruginosa. As shown in Figure 2B, P. chlororaphis could

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still grow using PCA as a sole carbon source, once protocatechuate 3,4-dioxygenase encoded

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by four different genes was deleted, the lack of growth on MM medium (+ PCA) indicated

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the participation of protocatechuate 3,4-dioxygenase in the degradation of PCA. As reported,

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PCA is metabolized to central metabolism via β-ketoadipate pathway

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Pseudomonas species degrade aromatic compounds via β-ketoadipate pathway

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the complete genome of P. chlororaphis HT66 was sequenced

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encoding the peripheral pathways for the catabolism of 4-hydroxybenzoate (pobA) and 5


33

24, 34

. Most of

24, 27

. Since

35

, we mapped the genes


112

benzoate (ben) in the chromosome.

113

The genes encoding phenol and PCA degradation were mapped in Figure 3 which include

114

two branches of the β-ketoadipate pathway. Sequence comparison indicated that P.

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chlororaphis HT66 contains genes encoding all of the enzymes involved in the two branches

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of the β-ketoadipate pathway and four genes encoding protocatechuate 3,4-dioxygenase

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participated in the catalytic degradation PCA. According to Figure 3B, 4-HBA transporter

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encoding by 4-hydroxybenzoate permease is located adjacent to the promoter, which may

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enhances the degradation of 4-HBA. MA is a naturally occurring intermediate in the

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degradation of 4-HBA of P. chlororaphis. The results indicated that the biosynthesis of MA

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could be achieved using the endogenous degradation pathways with high-efficient PobA from

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4-HBA.

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Genetic engineering to screen suitable locus for optimized expression of

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genes

125

P. chlororaphis HT66 is a high phenazine producing strain using the shikimate pathway.

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However, little plasmids availability hinders its in-depth study on molecular characterization

127

36, 37

128

heterologous gene overexpression, and the plasmids generally necessitate antibiotics for

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selection pressure, thus making the industrial bioprocess environmental-unfriendly and

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bio-incompatible. In addition, some plasmids were unstable to varying degrees and often lost

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in the later stage of fermentation 38. It is encouraging to achieve ideal industrial application

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through chromosomal integration, and multiple genes can be well regulated by different

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intensity promoters on chromosomes 39, 40. Recently, transcriptomics and proteomics analysis

. Therefore, chromosomal integration is preferred more than non-integrative plasmids in

6


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of different phenazine-producing strains revealed some candidates of high-expression genes

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with strong promoter

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phenazine synthesis pathway) and P. chlororaphis-AN-PHZ (with inactivated phenazine

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synthesis pathway) were used for transcriptome analysis. P. chlororaphis HT66 and P.

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chlororaphis P3 (a mutant with high phenazine production) were used for proteome analysis.

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The gene expression abundance was quantified by FPKM (Fragments per kilobase of exon

140

per million fragments mapped). The genes with expression abundance higher than phenazine

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synthetic genes in both strains were chosen as the candidates for overexpressing genes, and

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the phenazine synthetic genes were chosen for construction of heterologous pathway.

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Therefore, eGFP reporter gene encoding enhanced green fluorescent protein was used to

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assess all these candidates via substituting these genes

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different high expression genes were replaced by eGFP, and the activity of eGFP was then

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analyzed at a transcriptional and translational level to check the activity of promoters.

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As shown in Table S2, the phenazine biosynthesis genes transcription driven by the native

148

phenazine promoter (Pphz) was remarkably high; indicating that locus of a phenazine

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biosynthesis gene cluster is the candidate for exogenous gene cluster expression. The

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transcription of some genes (renamed as a, b, c, d, e, f) was quite high both in high and

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suppressed phenazine producing strains, indicating that these genes are driven by strong

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promoters that could be candidates for genes overexpression, and the expression of these

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genes are constitutive.

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We used eGFP to substitute each of these genes to reveal the expression of exogenous genes

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using these promoters. RT-PCR analysis revealed that the transcription of eGFP driven by Pf

12

as shown in Table S2. The P. chlororaphis-AN (with strengthened

41, 42

. Using eGFP as a reporter gene,

7



156

promoter (Pf) was considerably higher than those driven by Pa, Pb, Pe, Pd, Pc, and Pphz

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(Figure 4C). In the phenazine biosynthesis pathway, the transcription of genes near to

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promoters was greater than those far away from promoters. Once the phenazine production

159

was enhanced, the expression of these genes was improved. The fluorescence of each cell

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was also measured as displayed in Figure 4A and 4B. Comparing these promoters, the

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maximum fluorescence of each cell was detected at 60 h when driven by Pf, in accordance

162

with the results of RT-PCR. However, no obvious influence was observed on the cell growth

163

when genes a, b, c, d, e and f were substituted by eGFP (Figure S2A), the cell growth was

164

enhanced when phenazine biosynthesis genes were inactived (Figure S2B). Based on the

165

results, these genes are driven by stronger promoters that could be candidates for substitution

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with target genes.

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Construction of a synthetic pathway for 4-HBA in P. chlororaphis HT66

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4-HBA is usually produced in microbial metabolic pathways, therefore the tolerance of P.

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chlororaphis HT66 to 4-HBA was tested. Feeding different concentrations of 4-HBA (0-3 g/L)

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to the culture after 12 h of cultivation, the cell growth of P. chlororaphis HT66 was evaluated

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and results are shown in Figure S4. As the initial concentration of 4-HBA increased from 0 to

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2 g/L, no significant effect on the cell growth was detected, meanwhile, 4-HBA was

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metabolized dramatically in vivo. The inhibition effect increased in a dose-dependent manner,

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which displayed that a high concentration of 4-HBA reduced P. chlororaphis cell growth by

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26.7% (2.5 g/L) and 72.8% (3 g/L). Due to the characteristics, the tolerance of P.

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chlororaphis to 4-HBA was higher than E. coli in which 1 g/L 4-HBA reduced the cell

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growth by 25.7% 43. 8


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Three subfamilies of chorismatases have been reported to convert chorismate into different

179

(dihydro-) benzoate derivatives

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encoded chorismatease catalytic chorismate to form 4-HBA: ubic from E. coli, XanB2 from X.

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oryzae pv. oryzae, Rv2949c from M. tuberculosis, and sll1797 from C. Synechocystis sp.

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PCC6803. Each gene encoding chorismatease was used to replace the phzA or phzD in the

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chromosomes. First, ubic was used to determine which genome locus is the best based on

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phenazine biosynthesis pathway. Figure 5B shows that when pobA was deleted, only 5 mg/L

185

4-HBA was accumulated. When phzA was replaced by ubic, 433 ± 9.45 mg/L 4-HBA was

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produced, more than phzB (333 ± 9.457.14 mg/L 4-HBA), phzD (256 ± 1.58 mg/L 4-HBA),

187

phzE (83.73 ± 7.11 mg/L 4-HBA) and phzF (56.28 ± 2.34 mg/L 4-HBA) replacements

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(Figure 5A).

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Herein, 4 candidate genes were expressed on phzA locus individually. Figure 5B shows the

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amount of 4-HBA produced by each engineered strain after 96 h of fermentation. The titer of

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4-HBA produced by HT66-4X, carrying XanB2, was 1606 ± 28.08 mg/L and found to be the

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highest level of 4-HBA production among the four transformants. HT66-4U carrying ubic,

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HT66-4R carrying Rv2949c and HT66-4S carrying sll1797 produced 433 ± 9.45 mg/L, 93.72

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± 7.11 mg/L, and 56.28 ± 2.36 mg/L at the high titer, respectively. When XanB2 was used to

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substitute phzD under the control of Pphz (HT66-4XP), 1596 ± 32.08 mg/L 4-HBA was

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produced; it was similar to substitute phzA.

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Based on these results, one strain as chassis cell for the production of MA was constructed

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named HT66-MA with pcaG/H, pcaα/β, and catB deleted, and XanB2 inserted to the phzD locus

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in P. chlororaphis HT66.

44

. Therefore, we focused on the 4 candidate genes that

9



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Creation of a biosynthesis pathway for muconic acid from glycerol

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Many laboratories have described the engineering of strains for MA production using lots of

202

precursors

203

of pathway enzymes from self-replicating plasmids, which might cause a metabolic burden

204

on the host cell. In addition, this leads to the genetic instability, which makes these strains

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unsuitable for industrial applications

206

biosynthesis pathway with a β-ketoadipate pathway in P. chlororaphis, MA was

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biosynthesized instead of being plasmid and inducer dependent.

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Accordingly, 4-HBA has also been reported as a precursor in E. coli to produce up to 170

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mg/L MA 31. After successfully constructing the synthetic pathway for 4-HBA biosynthesis,

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the next step was to take advantages of 4-HBA degradation pathway for MA production.

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According to Figure 3C, the heterogeneous aroY from Kelbsiella pneumoniae, and

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endogenous pobA were genetically introduced into the MA-production strain HT66-MA to

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replace phzB, in order to guide the flux from 4-HBA to MA. According to Figure 1, phzE

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catalyzes

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phenazine-1-carboxylic acid or phenazine-1-carboxamide (PCN) was synthesized; therefore

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phzE was deleted yielding HT66-MA0 derivative strain which could not synthesize

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phenazines again. Figure 6B represents that HT66-MA0 produced 305 ± 24.46 mg/L MA

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after 60 h of fermentation. To confirm the MA production, a culture supernatant of HT-MA0

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was analyzed by UPLC/MS, where the specific peak was detected at about m/z of 141.02

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(negative ions) in Figure S3.

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To enhance the productivity of MA, exogenous catA from Acinetobacter sp. ADP1, aroY and

31, 45-48

. Nevertheless, these modifications were primarily based on the expression

chorismate

to

38, 49

. In this study, combining the ubiquinone

2-amino-2-desoxyisochorismic

10


acid

(ADIC),

and

then

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endogenous pobA were co-expressed with their own ribosome binding site (RBS) to

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substitute the phzA, phzB in chromosome using the phenazine promoter (Pphz), and derivative

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strain HT66-MA1 was constructed. When fermented in KB medium, 432 ± 13.06 mg/L MA

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was produced. To the best of our knowledge, this is the first report on metabolic engineering

226

of a foreign MA biosynthesis gene cluster integrated to the phenazine biosynthesis pathway

227

for MA biosynthesis based on the shikimate pathway of P. chlororaphis, with the yield of

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MA 24.2 mg/mg glycerol in the culture of HT-MA1. In comparison to traditional

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plasmid-based methods, combining endogenous ubiquinone biosynthesis pathway with

230

protocatechuate degradation pathway using chromosomal integration for MA production

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from glycerol has the advantages of cost-competitiveness, environmental friendliness and

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feedstock renewability.

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Modular optimization of the synthetic pathway of MA

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Multiple gene-promoter shuffling (MGPS) 50, artificial promoter library and mutant promoter

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library 51, 52 have been used to regulate the coordinated expression of multi-genes. Numerous

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works describing deregulation of the feedback inhibition, enrichment of the shikimate

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pathway, and increased supply of PEP and E4P, have been carried out to enhance the

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biosynthesis of target compounds from shikimate pathway

239

carbon flux from central carbon metabolism (CCM) into the shikimate pathway involves

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deregulation of feedback inhibition and transcription, as well as overexpression of critical

241

genes by genetic manipulation 28. Previous reports demonstrated that RpeA (encoded by rpeA)

242

negatively controlled the expression of phenazine gene cluster, which belongs to the

243

RpeA/RpeB two-component signal transduction system (TCST)

53-56

11


. Approaches to increase the

57

, and TCST systems are


244

beneficial for prokaryotes to interact with the environment through both sensing and response.

245

The global regulatory genes rsmE and lon can hinder the production of phenazines. In our

246

previous study, inactivating regulatory genes such as lon, rpeA and rsmE, the yield of

247

2-hydroxyphenazine was considerably enhanced (10-fold) 37. Based on the previous findings,

248

lon, rpeA, and rsmE were sequentially deleted in HT66-MA1, yielding HT66-MA2 strain, to

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verify the influence of these genes on the expression of Pphz. Fermentation profile showed

250

that 528 ± 35.68 mg/L MA was produced in HT66-MA2, which increased 21% in comparison

251

with HT66-MA1. In order to further enhance the yield of MA, the competitive pathway of

252

phenazine was evaluated by deleting phzE (HT66∆phzE and HT66-MA0). Figure 6A showed

253

that with phzE inactivation, the expression of all the genes of the competitive pathway from

254

chorismate was slightly increased, and no significant products accumulation was detected by

255

HPLC and UPLC-Q/TOF MS (data not shown), indicating that these competitive pathways

256

were weaker in P. chlororaphis HT66. Avoiding the auxotrophic strains in this study, it is

257

unnecessary to delete these competitive pathways. pykA and pykF, the genes encoding

258

pyruvate kinase to convert metabolic precursor PEP to TCA cycle, were both upregulated,

259

pykA increased 20-fold and pykF increased 4.5-fold. The gene pykA was inactivated and pykF

260

was retained to maintain the strain growth as normal, the derivative HT66-MA3 was obtained.

261

After fermentation and HPLC analyses, the production of MA of HT66-MA3 increased to

262

934 ± 22.00 mg/L.

263

Improvement of a rate-limiting step in MA production

264

To further enhance the MA production, we attempted to identify and improve the

265

rate-limiting step in the biosynthetic pathway of MA production. As reported previously in 12


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our study, six key genes (ppsA, tktA, phzC, aroB, aroD, and aroE) were overexpressed to

267

strengthen the shikimate pathway, and the target products increased significantly 37. The first

268

group included aroE, aroD, aroB, tktA and ppsA generally used to enhance the carbon flux to

269

chorismate. The second group included the genes required to increase the carbon flow from

270

chorismate to 4-HBA, XanB2 was adopted as the candidate. When feeding different

271

concentration of 4-HBA (0-2 g/L) in HT-MA3, almost all of 4-HBA was converted to MA

272

without 4-HBA (PCA or MC) accumulation after 48 h culture indicating that “4-HBA—MA”

273

is not a rate-limiting step in the whole biosynthesis pathway (Table 3). On phzE deletion, the

274

expression of 2-keto-3-deoxy-D-arabino-heptulosonate-7-phosphate synthase (phzC), a first

275

biosynthetic enzyme in shikimate pathway, was downregulated (Figure 6A). Therefore, the

276

third group included phzC and XanB2 genes. All the genes were separately cloned into

277

pBBR-MC2 and then transformed into HT-MA3 to obtain HT66-MA3-I, HT66-MA3-II and

278

HT66-MA3-III derivatives. Figure 6C portrayed that all groups introducing foreign plasmids

279

lead to delayed cell growth, but the expression of genes integrated into the genome locus was

280

superior to the plasmid-based expression strategy. It can be seen that overexpression of

281

aroE-aroD-aroB-tktA-ppsA had no significant effect on the enhancement of MA production

282

(Figure 6C). When overexpressed XanB2 was involved in the conversion of chorismate to

283

4-HBA, the MA concentration markedly increased with a titer of 1634 ± 116 mg/L as

284

compared to HT-MA3. The co-overexpression of phzC and XanB2 further increased the MA

285

production up to 1987 ± 97 mg/L compared with sole XanB2 overexpression. Herein, phzC

286

and XanB2 were chosen as the candidates for overexpression to enhance the MA production.

287

These critical genes were overexpressed via replacing the corresponding genes screened 13



288

according to Figure 4. Chorismatase XanB2 and promoter Pphz were co-expressed on phzD

289

locus under the control of Pphz to obtain HT66-MA4. XanB2 and phzC were co-expressed to

290

replace the gene f using the corresponding promoter to obtain HT66-MA5 (Figure 8). HPLC

291

analysis showed that 1645 ± 30.21 mg/L MA (in HT66-MA4) and 2375 ± 68.99 mg/L MA (in

292

HT66-MA5) were produced after 60 h of fermentation, with a yield of 91.4 and 131.9 mg/g

293

glycerol, respectively. Thompson et al. co-expressed the “DHS-MA” and “4-HBA-MA”

294

pathways in parallel to develop a synthetic “metabolic funnel”, enabling maximal net

295

precursor assimilation and flux while preserving native chorismate biosynthesis, and obtained

296

the production of MA up to 3.1 g/L at a yield of 158 mg/g glucose 48. According to the report,

297

the “metabolic funnel” strategy was used to co-express DHS-derived pathway to enhance the

298

production of MA.

299

Three genes, catA, aroY, and aroZ were cloned into gene a locus using Pa promoter and

300

generated derivative strain HT66-MA6. Notably, more than 2676 ± 68.15 mg/L MA was

301

produced, with a yield of 148.7 mg/g glycerol (Figure 6B). The amounts of 4-HBA and PCA

302

were also evaluated. PCA was not detected, whereas only 10 mg/L 4-HBA was detected,

303

maybe 4-HBA could not be catalyzed by pobA timely. Taken together, a synthetic pathway of

304

MA was constructed in P. chlororaphis HT66 combining the ubiquinone biosynthesis

305

pathway and PCA degradation pathway.

306

Improved MA production by feeding glycerol

307

Based on the glycerol utilization profile after 24 h culturing of HT66-MA6, no glycerol was

308

detected (Figure 7) at the initial concentration of 18 g/L, with the glycerol uptake rate 234.36

309

mg/L/h (0-24 h) and the highest MA production 2676 ± 68.14 mg/L. Therefore, fed-batch 14


Page 14 of 42

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310

cultures were carried out in shake flasks feeding 18 g/L glycerol every 24 h and culture

311

profiles of HT66-MA6 were depicted in Figure 7B. Remarkably, the amount of MA

312

production increased continuously up to 3376 ± 72.15 mg/L following glycerol feeding,

313

which represents the highest titer in P. chlororaphis. The present outputs were comparable

314

with those in recent reports, where the plasmid-based pathway was constructed for MA

315

synthesis, including “4-HBA-derived” pathway in E. coli (170 mg/L, shake flask)

316

“DHS-derived” pathway in E. coli (4 g/L, fed-batch)

317

flask)

318

“DHS-derived” pathway in E. coli produced up to 3.1 g/L MA at the expense of glucose

319

while introducing one auxotrophy

320

also used as endogenous precursors for MA biosynthesis in E. coli, with the titer of 389.96

321

mg/L, and 1.5 g/L, respectively.

322

At the end, HT66-MA6 was tested for the stability of the integrated genes. Our results

323

showed that MA productivity did not change after the strain was cultured for many

324

generations (Figure S5). Further, the tolerance of HT66-MA6 to MA was also tested. Using a

325

medium with different concentrations of MA (0-5 g/L), the cell growth of HT-MA6 was

326

evaluated. Based on the results, the specific growth rate (µ) in the cultures with 0, 1, 2, 3, 4, 5

327

g/L MA was 0.113 ± 0.003 h-1, 0.114 ± 0.011 h-1, 0.112 ± 0.009 h-1, 0.113 ± 0.010 h-1, 0.108 ±

328

0.006 h-1, 0.109 ± 0.008 h-1, respectively. These results suggested that P. chlororaphis HT66

329

was a suitable candidate host for MA production from glycerol independent of plasmid and

330

inducer. This is the first report on metabolic engineering of a plasmid-free foreign MA

331

biosynthesis in P. chlororaphis HT66 using glycerol as raw material. Though, Christopher et

59

, (559.5 mg/L, fed-batch)

60

58

31

,

and S. cerevisiae (141 mg/L, shake

. Plasmid-based coexpression of “4-HBA-derived” and

48

. As reported, anthranilate

15


61

and salicylic acid

30

were


332

al. have reported genome-integrated gene expression for MA production in P. putida KT2440

333

using exogenous tac promoter

334

comparable based on transcriptome and proteome analysis using native promoter. As glycerol

335

has a higher degree of reduction than glucose and produces more reducing equivalents to

336

form precursor PEP. In addition, as a by-product of biodiesel manufacturing process, the

337

utilization of glycerol has the advantages of cost-competitiveness and thus has a direct impact

338

on the economics of the industrial bioprocess. In this process, glycerol can be converted to

339

MA efficiently in the preferred fed-batch cultivation mode. Since MA was produced combing

340

the ubiquinone biosynthesis pathway and the protocatechuate degradation pathway in P.

341

chlororaphis, it might not be specific for other pathway utilization. Like as maleate or

342

fumarate can be biosynthesized in P. chlororaphis combining endogenous genamic acid

343

pathway and polyketide biosynthesis pathway. It is worth noting that using the chromosomal

344

integration of multiple genes or gene cluster can be stably expressed under different intensity

345

promoters on chromosomes without the selection pressure of plasmids.

25

. The present plasmid-free biosynthesis pathway was

346 347 348

16


Page 16 of 42

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349

Material and methods

350

Strains, plasmids, medium and culture conditions

351

All strains and plasmids used and developed in this study are listed in Table 2.

352

Oligonucleotide primers used are summarized in Table S1. Luria-Bertani (LB) medium

353

(Tryptone 10 g, Yeast extract 5 g, and NaCl 10 g, g/L) was used for incubating E. coli (37 °C)

354

and P. chlororaphis HT66 (CCTCC, M2013467) (28 °C) during the mutants construction.

355

King’s medium B (KB) (Glycerol 15 mL, Tryptone 20 g, MgSO4·7H2O 1.498 g, and

356

K2HPO4·3H2O 0.673 g, g/L) was used for P. chlororaphis fermentation. Minimal salt medium

357

(MM) (K2HPO4·3H2O 1.31 g, CaCl2 0.02 g, FeCl3 0.05 g, and MgSO4 0.02 g g/L),

358

MM-4-HBA (MM medium adding 1 g/L 4-HBA), or MM-PCA (MM medium adding 1 g/L

359

PCA) media were used as carbon source for P. chlororaphis. TB/SB (Tryptone 24, Yeast

360

Extract 48, KH2PO4 2.31, K2HPO4·3H2O 12.55, and Glycerol 25 g/L) medium was used for

361

protein expression. Where applicable, 100 µg/mL ampicillin, 50 µg/mL kanamycin, and 20

362

µg/mL gentamicin were added to the medium.

363

P. chlororaphis and its derivatives were activated at 28 °C overnight in KB agar media.

364

Single colonies from Petri plates were selected and then inoculated in 5 mL of KB broth in 50

365

mL flasks. Cultures were then incubated overnight at 28 °C with 200 rpm of shaking.

366

Portions of these cultures were then inoculated into 250 mL baffled flasks containing 60 mL

367

KB to achieve an initial OD600 of 0.02. The fermentation process was then initiated. After

368

12-96 h growth at 28 °C and 200 rpm, the samples were collected for the measurement of

369

target compounds and cell density. Triplicate experiments were carried out for each

370

fermentation test. 17



Page 18 of 42

371

Strain construction

372

All genes were PCR amplified with Q5 High-Fidelity DNA Polymerase (NEB) and each gene

373

was assembled with the respective plasmid using Gibson Assembly (NEB).

374

For pk18-phzAB construction, the 500 bp upstream of phzA and 500 bp downstream of phzB

375

were amplified by PCR using the genome of P. chlororaphis HT66 as a template. The

376

upstream and downstream fragments were cloned into the EcoR I and BamH I site. The

377

pk18-ubic-I, pk18-XanB2-I, pk18-Rv2949c-I, and pk18-sll1797-I plasmids were constructed

378

as follows; synthetic genes i.e. XanB2 from Xanthomonas oryzae pv. Oryzae, ubic from E.

379

coli, Rv2949c from Mycobacterium tuberculosis, and sll1797 from Cyanobacterium

380

synechocystis sp. PCC6803 were optimized for the P. chlororaphis codon usage, and then

381

cloned into EcoR I/BamH I site of pk18-phzAB. pk18-phzDE was constructed similarly to

382

pk18-phzAB, whereas pk18-ubic-I and pk18-XanB2-I were constructed when ubic and

383

XanB2 were cloned into EcoR I/BamH I site of pk18-phzDE individually.

384

The cassettes containing catA, pobA, and aroY, and containing phzC and XanB2 under the

385

control of endogenous Pphz promoter are referred as the MA module and 4-HBA module,

386

respectively. For pk18-MA module construction, catA from Acinetobacter sp. ADP1 and aroY

387

from Kelbsiella pneumoniae were optimized for the P. chlororaphis codon usage, and the

388

endogenous pobA gene was amplified using the genome as a template. Overlap PCR was

389

performed to align the three genes, which shared a 20 bp homologous region and the resulting

390

fusion fragments were cloned into EcoR I/BamH I site of pk18-phzAB.

391

For

392

aroE-aroD-aroB-phzC-tktA-ppsA cassette was amplified by PCR using the previous plasmid

pBBR-aroE-aroD-aroB-tktA-ppsA

plasmid

18


construction,

the

Page 19 of 42 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


393

15

394

For pBBR-XanB2-phzC construction, XanB2 was amplified using pk18-XanB2 as a template,

395

and phzC was amplified using genome as a template. After overlap PCR, the aligned

396

fragments were then cloned into EcoR I/BamH I site of pBBR.

397

Chromosomal in-frame deletions of the candidate genes were individually performed as

398

reported earlier 37. To substitute gene phzD with XanB2, a modified version of gene deletion

399

was used to amplify a 500 bp DNA fragment of phzD upstream, the opening read fragment

400

(ORF) of XanB2 and 500 bp DNA fragment of phzD downstream (Table S1). The fragments

401

were cloned into plasmid pk18mobsacB using NEBuilder HiFi DNA Assembly Master Mix.

402

In a similar way, other gene deletions or substitute derivative strains were constructed in the

403

corresponding strains.

404

Protein expression, purification, and in-vitro PobA enzyme assay

405

For protein expression, a single clone of BL21(DE3)-pobA strain was inoculated in 50 mL

406

TB/SB medium with 50 µg/mL kanamycin at 28 °C. Isopropyl β-D-1-thiogalactopyranoside

407

(IPTG) at a final concentration of 0.1 mM was added into the culture when OD600 reached to

408

0.6, followed by further growth at 16 °C for 16 h. After the designated time, the cells were

409

harvested and resuspended in 15 mL protein binding buffer and disrupted by ultrasonic

410

fragmentation. After centrifugation at 8000 g for 15 min, the supernatant was used as a crude

411

enzyme for enzymatic assays. The supernatant was purified by Ni-His affinity

412

chromatography and then confirmed by sodium dodecyl sulfate polyacrylamide gel

413

electrophoresis (SDS-PAGE). The concentration of total protein was determined by BCA

414

protein assay kit (TaKaRa) according to the manufacturer’s instructions. For crude enzymatic

as the template and the whole gene fragment was cloned into EcoR I/BamH I site of pBBR.

19



415

assays, different concentration of 4-HBA was added to 500 nM protein and the product was

416

determined by HPLC. The kinetic parameters of PobA were measured following the previous

417

method 33 with some modifications. Briefly, 1 mL reaction system contains 100 mM Tris-HCl

418

(pH 8.0), 10 mM FAD, 1000 mM NADPH, purified enzyme, and 0–1000 mM substrates

419

4-HBA. A 1000 mM substrate was used for specific activity detection, whereas 0, 25, 50, 100,

420

500, and 1000 mM substrates were used for kinetic parameters measurement. For PobA, the

421

enzyme concentrations were 100, 500, and 1000 nM, and the reactions were conducted at

422

28 °C for 5, 10, and 30 min. The kinetic parameters were calculated with OriginPro8.5

423

through non-linear regression of the Michaelis–Menten equation.

424

Identification of the catechol and protocatechuate branches of the

425

β-ketoadipate pathway in P. chlororaphis HT66

426

P. chlororaphis HT66 and its derivatives were cultured in LB medium for 20 h. After washing

427

and re-suspending with 5 µL phosphate-buffered saline (PBS), cells were inoculated in MM,

428

MM plus glycerol or 4-HBA (PCA) and the growth on plates was scored after two days.

429

Based on the reported β-ketoadipate pathway 62, genome-wide analysis of the located position

430

pobA, catA, pcaG/H, and pcaa/β was conducted according to the function of each gene. Further,

431

we mapped the genes encoding the peripheral pathways for the catabolism of

432

4-hydroxybenzoate (pobA) and benzoate (ben) in the P. chlororaphis HT66 chromosome.

433

HPLC and UPLC-Q/TOF MS analysis

434

Separation of 4-HBA, PCA, catechol, and MA were carried out using HPLC (Agilent

435

Technologies 1260 Infinity) with a C18 reversed-phase column (5 um, 4.6×12.5 mm). For

436

elution, water containing 0.1% formic acid (solvent A) and methanol containing 0.1% formic 20


Page 20 of 42

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437

acid (solvent B) were used as the mobile phases at a constant flow rate of 1 mL/min in a

438

gradient program. The gradient was initiated as a 95:5 mixture of A and B (0–2 min), shifted

439

to an 85:15 mixture of A and B (2–10 min), subsequently shifted to a 75:25 mixture of A and

440

B (10–20 min), and finally shifted to a 95:5 mixture of A and B (20–25 min). The product

441

concentrations (4-HBA, PCA, and MA) were determined using an ultraviolet absorbance

442

detector at 260 nm by injecting 20 µL sample. In all cases, external standards were prepared

443

and used to provide the calibrations for exact concentration determination.

444

The

445

spectrometry (UPLC-Q/TOF MS) was performed using a Primer UPLC-Q-TOF mass

446

spectrometer (Water Corp., Milford, MA, USA) equipped with an electrospray ionization

447

sources (ESI). UPLC was performed using a BEH C18 column (100 mm*21 mm, 1.71 µm) at

448

40 ℃. Mobile phase A (water containing 0.1% formic acid) and B (methanol containing 0.1%

449

formic acid) were used. The mass spectrometer was operated in the negative ESI mode, and

450

data acquisition was performed in selected-ion-monitoring (SIM) mode.

451

RT-PCR and quantitative real-time PCR

452

RNA preparation was performed using TaKaRa MiniBEST Universal RNA Extraction Kit.

453

The qPCR reactions were carried out in a 96-well plate in 10-µL reactions containing 5 µL 2×

454

SYBR green® Premix DimerEraser™ (Takara), 1 µL ROX reference dye, 1 µL cDNA sample,

455

and optimal concentrations of each primer. All samples were analyzed in triplicate. The

456

analysis of relative changes in gene expression from real-time quantitative PCR experiments

457

was based on 2-∆∆CT using rpod as a reference gene. The gene copy number of eGFP was

458

absolutely quantified according to a previous report 63.

ultra-high performance liquid chromatography-quadrupole

21


time-of-flight mass


459

ASSOCIATED CONTENT

460

S Supporting Information ○

461

The Supporting Information is available free of charge on the ACS Publication website at

462

DOI:

463

Main primer sequences used for plasmid construction; Transcriptome and proteome analysis;

464

Growth of P. chlororaphis HT66 in MM medium containing 0.6 g/L glycerol, 3-HBA,

465

4-HBA, PCA, 3,4,5-HBA, catechol, quinol as sole carbon source; Culture profiles of

466

candidate genes replaced by eGFP; Identification of 4-HBA and MA by LC-MC analyses;

467

Culture profiles of P. chlororaphis HT66 via feeding different concentration of 4-HBA after

468

culturing 12 h; The MA yield of the plasmid-free strain HT66-MA6 after subculture.

469

AUTHOR INFORMATION

470

Corresponding Author

471

* Tel: +86-21-3420-6742. Fax: +86-21-3420-6791. E-mail: [email protected]

472

(Xuehong Zhang).

473

OCRID

474

Songwei Wang: 0000-0002-2398-3105

475

Xuehong Zhang: 0000-0003-4894-0555

476

Author Contributions

477

S.W.W. and X.H.Z. conceived and designed the experiments. S.W.W. performed experiments,

478

analyzed the experimental data, and drafted the manuscript. M.B., and Y.N.Z., assisted in

479

experimental work and manuscript writing. H.B.H., and W.W. contributed reagents &

480

materials. X.H.Z. revised the manuscript. All authors contributed to the final paper. 22


Page 22 of 42

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481

Notes

482

The authors declare no competing financial interest.

483

ACKNOWLEDGMENTS

484

We are grateful to the Instrumental Analysis Center of Shanghai Jiao Tong University for

485

their skillful technical assistance in LC-MS analysis.

486

This work was supported by the National Natural Science Foundation of China (No.

487

31670033), and the National Key Basic Research Program of China (No. 2012CB721005).

488

ABBREVIATIONS

489

MA, muconic acid; 4-HBA, 4-hydroxybenzoic acid; PCA, protocatechuate; DHS,

490

3-dehydroshikimate; PEP, phosphoenolpyruvate; TCA, tricarboxylic acid cycle

491

23



492

References

493 494 495 496 497 498 499 500 501 502 503 504 505 506 507 508 509 510 511 512 513 514 515 516 517 518 519 520 521 522 523 524 525 526 527 528 529 530 531 532 533 534

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phenazine-producing

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Pseudomonas

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623 624 625 626 627 628 629 630 631 632 633 634 635 636 637 638 639 640 641 642 643 644 645 646 647 648 649 650 651 652 653 654 655 656 657


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658 659

27



660

Tables

661

Table 1. Specific activities and kinetic parameters of PobA Enzymes

PobA (P. chlororaphis) PobA (P. aeruginosa)

1

Page 28 of 42

U

Vmax

Km

kcat

kcat/Km

(U/mg protein)

(µM s-1)

(µM)

(s-1)

(µM s-1)

17.25 ± 1.5

0.36 ± 0.05

35.21 ± 4.12

15.24 ± 1.25

0.43

16.85 ± 1.39

0.35 ± 0.04

34.67 ± 9.51

14.12 ± 1.49

0.41

662

1

(2017) Rational engineering of p-hydroxybenzoate hydroxylase to enable efficient gallic acid synthesis via a novel artificial biosynthetic pathway 28


Page 29 of 42 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

663


Table 2. Strains and plasmids used in this study Strains S17-1 (λ pir)

Description

Source

E. coli res- pro mod+ integrated copy of RP4, mob+, used for incorporating constructs into P. chlororaphis

Lab stock

P. chlororaphis HT66

P. chlororaphis wild-type, PCN, Apr, Spr

Lab stock

HT66∆phzE

P. chlororaphis HT66 with phzE deleted

This study

HT66∆pobA

P. chlororaphis HT66 with pobA deleted

This study

HT66∆pcaa/β∆pcaG/H

P. chlororaphis HT66 with pcaa/β and pcaG/H deleted

This study

HT66, eGFP inserted to gene a (b, c, d, e, f) locus

This study

HT66-Ea (HT66-Eb, HT66-Ec, HT66-Ed, HT66-Ee, HT66-Ef) HT66-EA (HT66-EB, HT66-ED,

HT66, eGFP inserted to gene phzA

HT66-EE, HT66-EF, HT66-Ef)

(phzB, phzD, phzE, phzF, f) locus

HT66-4UA (HT66-4UB, HT66-4UD,

HT66, ubic inserted to gene phzA (phzB,phzD,phzE,phzF) locus

HT66-4UE, HT66-4UF, HT66-4UPD)

and phzD locus with Pphz

HT66-4U (HT66-4S, HT66-4R)

HT∆pobA, ubic (sll1797 or Rv2949c) inserted to phzA locus

This study

HT66-4X

HT∆pobA, XanB2 inserted to phzA locus

This study

HT66-4XPD

HT∆pobA, XanB2 inserted to phzD locus with Pphz

This study

HT66-MA

HT∆pcaG/H∆pcaa/в∆catB, XanB2 inserted to phzD locus

This study

This study

This study

HT66-MA, aroY inserted to phzA locus and pobA inserted to HT66-MA0

This study phzB locus with Pphz, with phzE deleted HT66-MA0, catA-pobA-aroY inserted to phzAB locus

HT66-MA1

This study under the control of Pphz

HT66-MA2

HT66-MA1∆pykA

This study

HT66-MA3

HT66-MA2∆lon∆rpeA∆remE

This study

HT66-MA3 I

HT66-MA3, harboring pBBR-aroE-aroD-aroB-tktA-ppsA

This study

HT66-MA3-II

HT66-MA3, harboring pBBR-XanB2

This study

HT66-MA3-III

HT66-MA3, harboring pBBR-XanB2-phzC

This study

HT66-MA3, co-expressed XanB2 and Pphz, XanB2 inserted to HT66-MA4

This study phzD locus, under the control of Pphz

29



Page 30 of 42

HT66-MA5

HT66-MA4, phzC-XanB2 inserted to f locus

This study

HT66-MA6

HT66-MA5, catA-aroY-aroZ inserted into a locus

This study

Plasmids

Description

Source

pk18mobsacB

Broad-host-range gene replacement vector, Kmr

Lab stock

pk18-pobA

pk18mobsacB containing pobA upstream and downstream, Kmr

This study

pk18mobsacB containing pcaG upstream and pcaH downstream, This study pk18-pcaG/H

Kmr pk18mobsacB

containing

XanB2,

phzD

upstream

and This study

pk18-XanB2

downstream with Pphz, Kmr pk18mobsacB containing catA-pobA-aroY, phzA upstream and This study

pk18-catA-pobA-aroY

phzB downstream, Kmr pk18mobsacB containing XanB2-phzC gene a upstream and This study

pk18-XanB2-phzC

downstream, Kmr pk18mobsacB containing catA-pobA-aroY, gene a upstream and This study

pk18-catA-aroZ-aroY

downstream, Kmr

pBBR-MC2

T7 and Lac expression vector, Kmr

Invitrogen

pBBR-XanB2

pBBR-MC2 containing XanB2, Kmr

This study

pBBR-aroE-aroD-aroB-tktA-ppsA

pBBR-MC2 containing aroE-aroD-aroB-tktA-ppsA, Kmr

This study

pBBR-XanB2-phzC

pBBR-MC2 containing XanB2-phzC, Kmr

This study

664 665

30


Page 31 of 42 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

666


Table 3. MA production and conversion rate via feeding 4-HBA in HT66-MA3 4-HBA (mg/L)

0

500

750

1000

1500

2000

MA (mg/L)

934.00

1430.29

1650.50

1768.60

2210.98

2585.68

98.74

96.76

90.10

89.24

86.42

Conversion rate

667

31



Page 32 of 42

668 669

Figure Captions

670

Figure 1. A schematic representation of modular engineering approaches in P. chlororaphis HT66

671

Gly3P: glycerol-3-phosphate; DHAP: dihydroxyacetone phosphate; GAP: glyceraldehyde 3-phosphate;

672

G6P,

673

3-dehydroshikimate acid; PCA(a), phenazine-1-carboxylic acid; PCN, phenazine-1-carboxamide; Phe,

674

L-phenylalanine; Tyr, Tyrosine; Trp, tryptophan. pykA pykF, pyruvate kinase; tktA, pyruvate synthase;

675

phzC, 2-keto-3-deoxy-D-arabino-heptulosonate-7-phosphate synthase; phzE, anthranilate synthase; XanB2,

676

chorismatase; aroY, protocatechuic acid decarboxylase; aroZ, 3-dehydroshikimate dehydratase; catA,

677

catechol 1,2-dioxygenase.

678

Figure 2. (A) Growth of P. chlororaphis HT66 and HT∆pobA in MM medium plus 4-HBA or glycerol as

679

sole carbon source; (B) Growth of P. chlororaphis HT66 and HT∆pcaG/H∆pcaα/β in MM medium plus PCA

680

or glycerol as sole carbon source (C) SDS-PAGE analysis of PobA protein; (D) PobA catalytic 4-HBA to

681

PCA; (E) The LC-MS of PCA.

682

WT, P. chlororaphis HT66; M1, strain HT66∆pobA; M2, strain HT66∆pcaG/H∆pcaα/β; MM, Minimal salt

683

medium.

684

Figure 3. The biosynthesis of catechol and protocatechuate via β–ketoadipate pathway and its regulation in

685

P. chlororaphis HT66. (A) Location of the gene clusters involved in degradation of benzoate and 4-HBA

686

on a line map of the chromosome; (B) Location of the gene clusters involved in degradation of 4-HBA on a

687

line map of the chromosome; (C) Predicted biosynthetic steps for the catechol and protocatechuate in P.

688

chlororaphis HT66. Inactivation of catB pcaG/H and pcaα/β is shown by “×”, and expression of aroY for

689

production muconic acid from 4-HBA is shown by the red arrow

690

Figure 4. Expression of eGFP on the candidate gene locus. (A) Fluorescence intensity per unit cell

691

expressed on a, b, c, d, e, f locus; (B) Fluorescence intensity per unit cell expressed on phzA, phzB, phzD,

692

phzE, phzF, phzG locus; (C) The copy number of eGFP expressed at a different locus

693

Figure 5. Production of 4-HBA based on the 4-HBA module. (A) The amount of produced 4-HBA based

694

on different gene locus using ubic; (B) The amount of produced 4-HBA based on different chorismatease

glucose

6-phosphate;

PEP,

phosphoenolpyruvate;

E4P,

32


erythrose

4-phosphate;

DHS,

Page 33 of 42 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


695

Figure 6. Culture profiles of various MA-producing transformants. (A) Relative fold change of the

696

chorismate competitive pathway; (B) MA production of different MA-producing transformants; (C) MA

697

production of different MA-producing transformants

698

Figure 7. Culture profiles of HT66-MA6. (A) Culturing HT66-MA6 at the initial glycerol concentration of

699

18 g/L; (B) Culturing HT66-MA6 by feeding 18 g/L glycerol per 24 h

700

Figure 8. A summary of the steps in the genetic and metabolic engineering of P. chlororaphis for muconic

701

acid production

702

33



703

Figures

704 705

Figure 1.

706 707 708 709 710 711 712 713 714 715 716 717 718 719 720 721

34


Page 34 of 42

Page 35 of 42 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

722


Figure 2.

723

724 725 726

35



727

Figure 3.

728 729

36


Page 36 of 42

Page 37 of 42

Figure 4.

Fluorescence/OD600 (a.u.)

730

4000

HT66

HT66-Ea

HT66-Ec

3000

(A)

HT66-Eb

HT66-Ed

HT66-Ee

HT66-Ef

2000 1000

100

0 0

12

24

36

48

60

72

84

96

Time (h)

Fluorescene/OD600(a.u.)

731 800 700 600 500 400 300 200

HT66-EG HT66-ED

HT66-EA HT66-EE

HT66-EB HT66-EF

(B)

75 50 25 0 0

12

24

36

48

60

72

84

96

Time (h)

732

log(copy numble/g (cDNA))

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


10.6

(C)

10.4 10.2 10.0 9.8 9.6 9.4 a

b

c

d

e

f phzA phzB phzC phzD

Samples 733 734 37



735

Figure 5.

Concentration of 4-HBA (mg/L)

736

600

HT66-4UF HT66-4UD

500

HT66-TUE HT66-4UPD

HT66-4UA HT66-4UB

(A)

400 300 200 100 0 0

20

40

60

80

100

Time (h)

737

Concentration of 4-HBA (mg/L)

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

Page 38 of 42

738

HT66-4X

2000 1600 1200 800 400

HT66DpobA

HT66-4R

HT66-4U

HT66-4XPD

HT66-4S

(B)

80 40 0 0

20

40

60

80

Time (h)

739

38


100

Page 39 of 42

Figure 6.

Relative fold change

740

(A)

HT66 HT66∆phzE HT-MA0

22.5 21.0 19.5 6.0 4.5 3.0 1.5 0.0 -1.5

phzA phzB phzC phzD ubic trpE pheA pabB pykA pykF ubic: 4-HBA--CoQ;

--

trpE: Anthranilate--L-Tryptophan

pabB: PABA--Folate ; pheA: L-Tyrosine/L-Phenylalnine pykA/pykF:pyruvate--TCA cycle

Concentration of MA (mg/L)

741

HT66-MA0 HT66-MA3

2500

HT66-MA2 HT66-MA4

(B)

1500 1000 500 0 0

20

40

60

80

100

Time (h) 0 2000

20h

40h

60h

80h

(C)

— — — — DCW

15

MA (mg/L)

12

1500 9

1000

6

500 0

3

A -M 66 T H

3

I -II 3 -II A3 A3 MA M M 666 6 6 T 6 H HT HT

39


0

Biomass, DCW (g/L)

742

743 744

HT66-MA1 HT66-MA5

2000


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



(A)

3000 2400

15

1800 10 1200 5

600

0 0

20

40

60

80

0 100

Time (h) 20 18 16 14 12 10 8 6 4 2 0

(B)

4000

3000

2000

1000

0 0

20

40

60

Time (h)

80

747 748

40


100


20


746

Figure 7.

Concentration of glycerol (g/L)

745

Concentration of glycerol (g/L)

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

Page 40 of 42

Page 41 of 42 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

749


Figure 8.

750 751 752

41



753

Graphical abstract

754

42


Page 42 of 42

Development of a Plasmid-Free Biosynthetic Pathway for Enhanced

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