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Microbial Synthesis of 5-Aminolevulinic Acid (ALA) and Its Co-production with Polyhydroxybutyrate (PHB) Tian Li, Ying-Ying Guo, Guan-Qing Qiao, and Guo-Qiang Chen ACS Synth. Biol., Just Accepted Manuscript • DOI: 10.1021/acssynbio.6b00105 • Publication Date (Web): 29 May 2016 Downloaded from http://pubs.acs.org on June 7, 2016

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ACS Synthetic Biology

Original paper (sb-2016-00105a) for the Special Issue dedicated to “Synthetic Biology in Asia”

Title:

Microbial Synthesis of 5-Aminolevulinic Acid (ALA) and Its Co-production with Polyhydroxybutyrate (PHB)

Authors:

Tian Li1, Ying-Ying Guo1,2, Guan-Qing Qiao1, Guo-Qiang Chen*1,2,3

Affiliations: 1

Peking-Tsinghua Center for Life Sciences, School of Life Science, Tsinghua

University, Beijing 100084, China 2

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

China 3

MOE Key Lab of Industrial Biocatalysis, Tsinghua University, Beijing 100081,

China

*Correspondence: Guo-Qiang CHEN School of Life Science, Tsinghua University, Beijing 100084, China Tel: +86-10-62783844, Fax: +86-10-62794217, e-mail: [email protected]

1

ACS Paragon Plus Environment


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ABSTRACT

5-Aminolevulinic acid (ALA), an important cell metabolic intermediate useful for cancer treatments or plant growth regulator, was produced by recombinant Escherichia coli expressing the codon optimized mitochondrial 5-aminolevulinic acid synthase (EC: 2.3.1.37，hem1) from Saccharomyces cerevisiae controlled via the plasmid encoding T7 expression system with a T7 RNA polymerase. When a more efficient auto-induced expression approach free of IPTG was applied, the recombinant containing antibiotic free stabilized plasmid was able to produce 3.6 g/L extracellular ALA in shake flask studies under optimized temperature. A recombinant E. coli expressing synthesis pathways of poly-3-hydroxybutyrate (PHB) and ALA resulted in co-production of 43% PHB in the cell dry weights and 1.6 g/L extracellular ALA, leading to further reduction on ALA cost as two products were harvested both intracellularly and extracellularly. This was the first study on co-production of extracellular ALA and intracellular PHB for improving bioprocessing efficiency. The cost of ALA production could be further reduced by employing a Halomonas spp. TD01 able to grow and produce ALA and PHB under continuous and unsterile conditions even though ALA had the highest titer of only 0.7 g/L at the present time.

Keywords 5-Aminolevulinic acid, PHB, Escherichia coli, Saccharomyces cerevisiae, Halomonas, T7 RNA polymerase, synthetic biology 2


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Introduction 5-Aminolevulinic acid (ALA) has potentials to be used as a photodynamic anti-cancer medicine

1-3

, a biodegradable photo-activated pesticide4,5, a potential plant growth

regulator6 especially under stress conditions7,8, and a key precursor of the cytochrome c, heme and vitamin B12 bio-synthesis9. Chemical synthesis of ALA is complicated with low yields and thus high price4. Biosynthesis of ALA has thus become attractive. Two alternative ALA biosynthesis pathways are reported in nature: the C5 pathway was found in higher plants, in algae and many bacteria including E. coli, involving three steps from glutamate, and the C4 pathway found in mammalian, yeast, and purple non-sulfur phototrophic bacteria, which condenses succinyl-CoA and glycine catalyzed by 5-aminolevulinic acid synthase (ALAS)4.

As ALA receives more and more commercial attention, its biosynthesis opportunity has been attracted increasing attentions these years5,9-16. Significant efforts have been made to improve its biosynthesis yield. For example, it was reported that some highly efficient foreign prokaryotic ALA synthesis genes were found and successfully expressed in recombinant E. coli for enhanced ALA synthesis, including hemA and hemO from Rhodopseudomonas palustris13,14, hemA from Rhodobacter sphaeroides17 or Agrobacterium radiobacter15,16, and the C5 pathway hemA gene, encoding a glutamyl-tRNA reductase fermentation processes

from Salmonella arizona12. Optimization

on the

were also conducted under batch and fed-batch culture

conditions11,16,17, leading to 7.30 g/L or 56 mM ALA in a 15-L fermenter. Some efforts

3



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to regulate ALA relevant or downstream pathways were also made for improving ALA production, including reduction on the activity of ALA dehydratase via addition of D-glucose18, the use of ALA dehydratase deficient mutant19, or most recently, regulation on the heme biosynthesis pathway. These efforts improved the ALA yield from 1.82 to 3.25 g/l in a 3-L bioreactor20. ALA bio-production has thus become more promising for industrial production.

Instead of using prokaryotic ALA biosynthesis genes reported by all other studies for ALA biosynthesis, in this study, a mitochondrial ALA synthase (EC:2.3.1.37) coding gene hem1 from eukaryotic Saccharomyces cerevisiae ATCC 204508 30,31, was used to construct recombinant E. coli strains under conditions of codon optimization and co-expression of T7 promoter system to further enhance ALA production. More efforts were also made to reduce ALA production cost by developing both antibiotic- and IPTG-free auto-induction expression systems.

Polyhydroxybutyrate (PHB), biodegradable polyesters synthesized by a wide variety of bacteria as carbon and energy storage inclusions, have been considered as environmentally friendly bioplastics also with many other promising applications21-25. PHB has however not yet found a market for bulk applications due to the high cost of production, which largely restricts PHB from broad commercial usage25. Since PHB exists as solid granules inside the cells, the extracellular culture broth is less useful and must be discarded after cell separation. It undesirably causes a waste of water during the manufacturing process. Therefore, a seawater based biotechnology had been

4


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studied to deal with this water problem26-29.

To improve the economy of PHB production, intracellular PHB production can be integrated with extracellular production of soluble and high value-added chemical ALA. The combination of these two production processes could not only lower the PHB production cost but also allow production of a second product from one fermentation process (Fig. 1). In addition, recombinant Halomonas TD which can be grown under continuous and unsterile conditions were exploited for both low cost ALA and PHB co-production.

Results and Discussion ALA production by E. coli expressing yeast mitochondrial ALA synthase

A synthetic ALA production pathway, namely, the C4 pathway, was used in engineered strains for ALA synthesis via the condensation of succinyl-CoA with glycine by ALA synthase (Fig. 1, Table I, Fig. S1). Previous researchers mostly focused on the prokaryotic ALA biosynthesis genes, especially the genes from photosynthetic bacteria like Rhodobacter sphaeroides17, but seldom take the eukaryotic ALA biosynthesis genes into consideration, which in fact, has been indicated promising in some earlier studies.30,31

Here, Saccharomyces cerevisiae mitochondrial ALA synthase (EC: 2.3.1.37) coding gene hem1 and

Rhodobacter sphaeroides ALA synthase coding gene hemA were

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cloned and expressed in E. coli, respectively, using plasmid pET-28a equipped with a T7 system. To maximize the heterologous ALAS expression in E. coli, whole sequence codon optimization was conducted based on the “one amino acid–one codon” approach 32 according to E. coli tRNA bias pool. The expression of codon optimized hem1 in E. coli LTT02 strain showed the best ALA synthetic capacity, and increased ALA production to 940 ± 42 mg/L, a nearly 3-fold enhancement compared to the wild type hem1 expression (Fig 3a). The expression level of codon optimized hem1 was also increased more than the wild-type (Fig. S2).

Enhanced ALA production by expressing codon optimized hem1 in various recombinant E. coli

As there are 3 predicted disulfide bonds in 548aa containing ALA synthase (according to predicting software http://clavius.bc.edu/~clotelab/DiANNA/), to facilitate the formation of these disulfide bonds in this recombinant protein in proper order, commercially available Origami E. coli B (DE3), a modified strain of thioredoxin reductase (trxB) and glutathione reductase (gor) genes mutant33-35 was used to produce ALA and was designated as strain LTT03, which was expected to improve ALA synthase activity. Shake flasks studies clearly showed that E. coli LTT03 strain increased ALA production to 1,609 ± 84 mg/L (Fig. 3b).

Strain LTT05 contains hem1 gene jointly over-expressed with T7 RNA polymerase under T7 promoter for strengthening the T7 system. The commonly used T7 expression system allows high-level expression from the strong T7 promoter, but it is 6


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restricted to limited T7 expression hosts, such as E. coli DE3 strains, which carries one copy of the phage T7 RNA polymerase gene on the chromosome under the control of a relatively weak lac promoter. In this work, we further enhanced the T7 system by constructing T7 RNA polymerase gene on the plasmid right after the codon optimized hem1 under the T7 promoter (Fig. 4a). Excitedly, we found that this approach significantly improved the extracellular ALA accumulation to 2,013 ± 113 mg/L despite the relatively poor cell growth, which accentuated its high efficiency and potential (Fig. 3b).

To improve ALA excretion, E. coli strains LTT06 and LTT07 were constructed both expressing an inner membrane transporter RhtA (rhtA) first known to be responsible for threonine and homoserine efflux transport, while later reported to accelerate the export of ALA12,36. The LTT06 strain expressed RhtA as a protein fused with the fluorescent mEos allowing detections of its location on the cell membrane (Fig. S3). While the LTT07 expressed RhtA linked with the optimized hem1 to assist the ALA synthase anchoring on the cell membrane near to the ALA exporter, so that ALA could be conveniently excreted out of the cells (Fig. 3b).

Strain LTT10, a cell wall weakened E. coli JM109SG5△ with its five cell wall synthesis relating genes (PBPs1b, PBPs5, AmpC, AmpH ,ddlB ) deleted, on the other hand, was engineered to express the optimized hem1 for possible easy ALA leakage out of the cells (Fig. S4). However, it turned out that the extracellular ALA accumulation actually decreased, possibly related to poor cell growth (Fig. 3b).

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E. coli strain LTT16 was engineered to express ALA synthase and Vitreoscilla hemoglobin (VHb) encoded by gene vgb

37-38

for more ALA accumulation. Heme, a

downstream metabolite of ALA, was reported to be a feedback repressor of ALA synthase42 (Fig. 1), down-regulating the ALA biosynthesis process. A number of previous studies indicated that heme and its direct precursor play an important role for functional expression of some hemoproteins like VHb

39,40,41

. Having heme as a

cofactor, hemoproteins like VHb may help to uptake heme which releases some down regulation effect on ALAS.

Results showed an increase to 1,180 ± 86 mg/L in shake-flask studies expressing VHb compared with only 940 ± 42 mg/L produced by the control (Fig. 3b). Thus, there is reason to believe that expression of VHb benefits ALA accumulation by helping ALA synthase to uptake some feedback repressor heme.

Among these candidate recombinant E. coli strains, LTT05 with enhanced T7 system achieved the highest ALA yield. Co-expression of T7 RNA polymerase largely increased the expression level of codon optimized hem1 (Fig. S5). This is also the first study on using the T7 RNA polymerase co-expressed enhanced T7 system for ALA biosynthesis.

In addition, plasmid pLTT05- H1T7P-C from LTT05 was transformed to 9 commonly used E. coli hosts to evaluate the different ALA synthesis abilities. The best expression platform was found to be the E. coli BL21 (Fig. 3c).

8


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ALA production by E. coli under a T7 enhanced and antibiotic-free system

The addition of antibiotics into the fermentation broth to stabilize plasmids results in not only inconvenience for industrial production but also cost disadvantages as well as difficult waste water treatments. Thus, we further engineered the strain LTT09 based on LTT05. An antibiotic free stable system was established by deleting two essential genes ispH and folK, on the E. coli genome and re-locating them to the plasmid43 (Fig. 4a).

Results demonstrated that the addictive recombinant E. coli LTT09 could be used for long lasting fermentation processes avoiding the use of antibiotics while maintaining ALA accumulation (Fig. 4b), its plasmid stability and ALA production was much better than LTT05 cultivated without antibiotic (Fig. S6). Even though ALA production (1,725 ± 81 mg/L) by the LTT09 antibiotic-free system was not that high due to limitations of the addictive system: the plasmid copy number is controlled by the host requirement for essential gene expression not by the high copy number plasmid itself 43.

This study showed that strain LTT09 containing an antibiotic-free system for long lasting ALA production was feasible.

ALA production by an auto-induction (IPTG-free) system under better growth conditions

ALA is unstable under elevated temperature and high pH

44

, a better pH control

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obtained from a gradient optimization of the phosphate buffer resulted in improved ALA productivity observed in a 3-fold common buffer solution (K2HPO4·3H2O 83.4 mM and KH2PO4 216.6 mM) (Fig. 5a).

The T7 system is commonly used in most of the important studies requiring strong induction by expensive IPTG (isopropyl β-D-1thiogalactopyranoside) including ALA synthesis. To avoid use of expensive IPTG induction, auto-induction was developed as a convenient way to perform recombinant protein production in small laboratory scale for lac operon-controlled systems45,46,47.

Auto-induction is commonly employed for lab scale protein production

46-48

yet

seldom used for other purposes including ALA biosynthesis. The auto-induction expression system provides several advantages over the traditional IPTG-induction method including growth without process intervention as inductions occur at an appropriate cell density under metabolic controls of the expression host; no need to monitor cell growth in a log-phase culture; glucose presence repressing the expression leakage; possibility of toxic protein production49; wider range of growth temperatures of 18°C to 37°C45. These advantages are welcomed for production of ALA which is unstable at a higher temperature44.

Effects of induction times in conventional LB and TB media together with the auto-induction were studied combined with optimizing the growth temperature in the auto-induction system. Results indicated that ALA production was significantly influenced by the induction time. ALA synthesis was improved to 2,745 ± 85 mg/L, 10


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demonstrating that auto-induction was not only a low-cost option, but also highly effective for ALA production (Fig. 5b)

The highest ALA production was found at 25°C based on temperature optimization studies (Fig. 5c), although the expression level of hem1 at 25°C was not the highest (Fig. S7). The auto-induction system was found to be most effective at 25°C under a stable pH controlled using a 3-fold common buffer concentration (K2HPO4·3H2O 83.4 mM, KH2PO4 216.6 mM).

Co-production of ALA and poly (3-hydroxybutyrate) or PHB by recombinant E. coli

A recombinant E. coli LTT19 PHB+ALA+ harboring double plasmids capable of co-producing ALA and PHB was constructed (Fig. 1, Fig. 6a) using E. coli BL21 as a host. As single product control strains, recombinant E. coli LTT19 PHB-ALA+ and E. coli LTT19 PHB+ALA- were also constructed (Table I). E. coli LTT19 PHB+ALA+ produced 1,562 ± 51 mg/L ALA and 43 ± 2 wt% PHB simultaneously when grown in the shake flasks, slightly lower than their individual production controls producing 1,751 ± 58 mg/L ALA and 47 ± 4 wt% PHB, respectively. Further optimization should lead to much better results (Fig. 6b).

Co-production of ALA and PHB can solve the problem of wastes. Since PHB exists as granules inside the cells, the supernatant is less useful and must be discarded after biomass separation. In contrast, ALA is accumulated extracellularly and the cells are

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useless. The co-production can make both the cells and supernatant useful (Fig. 6a).

In addition, co-production of ALA and PHB allows production of two products from one fermentation process, these two products were easy separated, which also save time and energy.

Halomonas TD as a low-cost platform for co-production of ALA and PHB

Halomonas TD01 was exploited as a novel ALA and PHB co-production platform by constructing two recombinant Halomonas TD01 strains including LTT21 harboring pTDALA01 (pRE112-pMB1 inserted with hem1) and LTT22 containing pTDALA02 (pSEVA341-Pporin inserted with hem1). Co-production of ALA and PHB was clearly observed in recombinant LTT22. The recombinant strain grown in shake flasks produced 635 ± 24 mg/L ALA extracellularly and 22 ± 3 wt% PHB intracellularly (Fig. 7).

Results indicated that Halomonas TD01 did have the potential to be further developed to a novel low-cost platform for ALA and PHB co-production. As Halomonas TD can be grown under unsterile and continuous conditions favorable for fermentation industry, and can produce PHB without additional molecular engineering26-29, Halomonas TD01 could be a better low-cost platform for ALA and PHB co-production compared with E. coli.

ALA has been well investigated to enhance the salinity stress tolerance of plants6,7,50. Recently salt tolerant ALA producing purple non-sulfur bacteria was studied for 12


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agricultural application but the ALA yield was too low51,52. Therefore, Halomonas is quite promising to meet demands as a stronger ALA producer while also tolerates the saline-alkali environments. In addition, intracellular PHB can further increase stress resistance of bacteria21, 53, and may benefit the plants by helping them resist the injurious effects of salt stress of plants grown in salty environments.

Conclusions In this study, we reported the expression of novel codon optimized mitochondrial ALA synthase from eukaryotic Saccharomyces cerevisiae in Escherichia coli, and engineered a series of recombinant Escherichia coli strains using different methods to further optimize the expression and improved the ALA production. Among them, the recombinant LTT05 with enhanced T7 expression system via co-expression with T7 RNA polymerase showed the highest ALA production ability. The recombinant LTT05 was further improved with an auto-induction system to reduce the ALA production cost avoiding IPTG induction, resulting in 3,584 ± 165 mg/L extracellular ALA produced in shake flasks at 25oC. To further reduce ALA production cost, extracellular ALA production was linked with intracellular bioplastic PHB

synthesis in

recombinant E. coli LTT05 containing PHB synthesis operon, the resulting strain termed E. coli LTT19 PHB+ALA+ produced 1,562 ± 51 mg/L ALA and 43 ± 2 wt% PHB when grown in shake flasks for 48 h. To further reduce the ALA production cost, a halophile Halomonas strain TD able to grow under continuous and unsterile

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conditions was engineered to co-produce ALA and PHB. The successful exploration of multiple engineering approaches has proven effective for improving the production of the value added chemical ALA. Future scale-up efforts should demonstrate the economic advantages of the co-production technology.

Methods

Bacterial Strains and Plasmids

All the microorganisms and plasmids used in this study are provided in Table I. Molecular cloning standard procedures including vector isolation, DNA amplification, restriction enzyme digestion as well as other DNA manipulations were employed for plasmids construction. DNA purification and plasmids isolation kits were purchased from Biomed (Beijing, China). Restriction enzymes and ligation kits were supplied by Thermo (Beijing, China). Taq and Pfu DNA polymerase used in this study were from TransGen (Beijing, China). Gibson assembly was conducted following the published paper54, and Gibson Assembly® Master Mix was purchased from New England Biolabs (Beijing, China)

The PCR primers used in this study are provided in Table S1. Primers were synthesized by Invitrogen (Shanghai, China). And gene sequencing were conducted by AuGCT Biotech (Beijing, China). All the plasmids constructed were verified by PCR and by DNA sequencing to confirm the correctness of the constructs. The wild type 14


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hem1 gene was amplified using polymerase chain reaction (PCR) using the genome of Saccharomyces cerevisiae as DNA template, and the codon-optimized hem1 gene (sequence shown in Supplementary) was synthesized by Qinglan (Shanghai, China). The T7 RNA polymerase gene was amplified from E. coli BL21 (DE3) strains.

E. coli Trans1-T1 strain was used as the host strain for plasmids construction. E. coli BL21 (DE3) strains were used for gene expression under normal T7 expression system. Through all the construction process, all E. coli strains were cultivated in Luria-Bertani (LB) medium (10 g/L tryptone, 5 g/L yeast extract and 10 g/L NaCl).

Construction of essential gene containing plasmids and study on plasmid stability

E. coli JM109SG was used as the start strain for further genetic manipulations43. Essential gene ispH and folK were amplified from the genome of E. coli JM109 by primers HH-GF, HH-GR, KK-GF, KK-GR and the fragments were subsequently inserted

into

hem1

and

T7

RNA polymerase

operon

containing

vector

pLTT09-H1HKT7P, plasmid maps of the constructed plasmids were shown in Supplementary Fig. S1.

To investigate the stability of plasmids, cells were cultivated in LB medium at 37℃ for 24 h, and were re-inoculated into a new LB medium for another 24 h. After 8 rounds of re-inoculations, the resulting cell culture was diluted 10-5 folds and spread onto LB agar plates with or without chloromycetin. The LB plates were cultivated for 18 h at 37℃. Subsequently, colony-forming units (CFU) were counted and compared

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for the two types of LB agar plates43.

Construction of recombinant Halomonas TD01

Halomonas TD01, isolated from Aydingol Lake of Xinjiang Province in China, was deposited in CGMCC (China General Microbiological Culture Collection Center, Beijing) with a collection No. 4353. Its wild type was used as a start strain for genetic manipulations. E. coli S17-1 was used as a vector donor for conjugation. E. coli and Halomonas spp. TD01 and their derivatives were cultivated in LB medium and 60-LB medium, respectively. 60-LB medium contains (g/L): NaCl 60, tryptone 10, yeast extract 5. All bacterial strains and plasmids used are listed in Table I, plasmid maps of the constructed plasmids were shown in Supplementary Fig. S1. Conjugation was found to be an effective method for transforming plasmids into Halomonas TD0150,51. The donor cells were incubated overnight while recipient ones were incubated in the presence of relevant antibiotics to an OD600=0.6–0.8. Cells were harvested at 5000 g, 4℃ for 10 min, then washed twice with LB (for E. coli S17-1) or 60-LB (for Halomonas TD strains), followed by mixing at a 1:1 ratio, subsequently placed on 60-LB plates at 37℃ for 6 h. Then cells were re-suspended and incubated for 8–12 h at 37℃ in 60-LB agar plates containing chloromycetin.

Shake flask studies on ALA accumulation

The Luria-Bertani (LB) medium was used for all seed culture. These were incubated for 8 h at 200 rpm, 37oC in shake flasks containing 40 ml LB medium. The

16


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fermentation was conducted in 500 ml shake flasks containing 100 ml adjusted LB, TB, and ZYP 5052 (auto-induction) media with phosphate buffer solution. The adjusted LB medium contains: tryptone 10 g/L, yeast extract 5 g/L, 1-7 fold phosphate buffer (1-fold buffer: K2HPO4·3H2O 27.8 mM, KH2PO4 72.2 mM) ; the adjusted TB medium contains: tryptone 12 g/L, yeast extract 24 g/L, 1-7 fold phosphate buffer ; The adjusted ZYP 5052 (auto-induction) medium for auto-induction system contains: ZY (N-Z-amine AS 10 g/L, yeast extract 5 g/L), 1 mM MgSO4, 5052 ( 0.5 % glycerol, 0.05% glucose, 0.2% α-lactose ), 1-7 fold phosphate buffer. For Halomonas TD01, both seed and fermentation cultures are 60-LB medium, No IPTG and phosphate buffer were added.

For ALA accumulation, two-step shake-flask cultivation was conducted. First, the shake flasks were incubated at 200 rpm and 37℃, then the mixture was cooled down until

the

end

of

process.

In

the

adjusted

LB

or

TB

medium,

isopropyl-β-D-thiogalactopyranoside (IPTG) was added to induce the expression of ALAS at the final concentration of 0.05 mM. The initial conditions for the ALA production were 3.0 g/L glycine, 6.0 g/L succinic acid, and 2.0-8.0 h after incubation, IPTG was added and culture temperature was adjusted to 16, 20, 25, 30, or 37°C. The pH was controlled by a 1-7 fold phosphate buffer. Additional 10.0 g/L glucose, as well as 5.0 g/l glycine and 10.0 g/l succinic acid were added at 18 h. The cells were harvested after cultivation for 48 h. Antibiotics including (µg/ml) 100 kanamycin, 50 ampicillin, 35 chloramphenicol were added, respectively or in various combinations. High phosphate concentration induces kanamycin resistance, kanamycin concentration 17



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was elevated accordingly45.

Co-production of ALA and PHB by recombinant E. coli and Halomonas TD01.

To coproduce ALA and PHB, Luria-Bertani (LB) medium was used for recombinant E. coli seed cultures that were incubated for 8 h at 200 rpm and at 37°C in shake flasks containing 40 ml LB medium. The growth was conducted in 500-ml shake flasks containing 100 mL adjusted LB medium: tryptone 10 g/L, yeast extract 5 g/L, 3-fold phosphate buffer (K2HPO4·3H2O 83.4 mM, KH2PO4 216.6 mM). For the co-production process, two-step shake-flask cultivation was conducted. The initial conditions were (g/L) 3.0 glycine, 6.0 succinic acid, and IPTG and 20 g/L glucose was added 4.0 h after incubation. Culture temperature was adjusted to 30°C. Additional 10.0 g/L glucose, as well as 5.0 g/L glycine, 10.0 g/L succinic acid were added at 18 h. Cells were harvested after cultivation for 48 h.

To grow Halomonas TD strains, LB medium containing 30 g/L NaCl was used. 1.0 g/L glycine and 2.0 g/L succinic acid were added initially. 8 h after incubation, 20 g/L glucose was added and culture temperature was adjusted to 30°C. Additional 10.0 g/L glucose, 2.0 g/L glycine, 4.0 g/L succinic acid was added at 18 h. Cells were harvested after cultivation for 48 h.

Analytical Method

The production of ALA was analyzed using the Modified Ehrlich’s Reagent after the cultures were centrifuged, following the method described by Mauzerall and 18


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Granick55.

For quantifying of PHB contents, cells were harvested by centrifugation at 10,000 g for 10 min, then washed with distilled water and centrifuged again. Cell dry weights (CDW) were measured after the lyophilization of cell pellets. PHB contents as well as compositions were analyzed via a gas chromatograph (GC-2014, SHIMADZU, Japan) after methanolysis26. Pure PHB was used as standard and quantified as monomeric 3-hydroxybutyric acid methyl ester (3HB).

For monitoring microbial growth, optical densities (OD) of cell suspensions were measured at a wavelength of 600 nm with a UV spectrophotometer (Biowave DNA, Biochrom, England).

Author Contributions T.L. and Y.Y.G. contributed equally to this work. T.L. and Y.Y.G. and G.Q.C.designed the project. T.L. and Y.Y.G. performed the experiments and T.L. prepared the draft paper. G.Q.C. supervised the study.

Notes

The authors declare no competing financial interest.

Acknowledgement This research was financially supported by 973 Basic Research Fund (Grant No.

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Page 20 of 41

2012CB725201) and a Grant from National Natural Science Foundation of China (Grant No. 31430003). Xiaoran Jiang contributed the construction of E. coli JM109SG5△.

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Supporting Information Figure S1. Plasmids constructed in this study. Figure S2. Expression levels of hem1 in E. coli LTT01 and LTT02 Figure S3. Location of fusion proteins RhtA and mEos, respectively Figure S4. Evidence of E. coli JM109SG5delta gene deletions. Figure S5. Expression levels of codon optimized hem1 in E. coli LTT02 and LTT05. Figure S6. Comparison of plasmid stability and ALA production by E. coli LTT05 and LTT09. Figure S7. Expression levels of hem1 under different temperatures. Figure S8. Co-production of ALA and PHB by recombinant E. coli and Halomonas TD. Table S1. Oligonucleotide primers used in this study. Table S2. Comparison of the specific ALA production rates of some recent studies. Table S3. Sequence of codon optimized hem1. Methods for RT-PCR, JM109SG5delta construction.

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Table I

Bacterial strains and plasmids used in this study

Strains or Plasmids

Description

Reference/source

E.coli Origami B (DE3)

F- ompT hsd SB( r B- mB-) gal dcm lac Y1 ahpC (DE3) gor 522::Tn10 trxB (KanR，TetR)

TransGen Biotech Co., Ltd.

E. coli MG1655gyyEF

F- lambda- ilvG- rfb-50 rph-1 ∆metE ∆hemF

Guo et al this lab

A vector donor in conjugation, harbors the tra genes of

26

Strains

E. coli S17-1 E.coli JM109SGIK E.coli JM109SG5△

plasmid RP4 in the chromosome; proA, thi-1 43

E.coli JM109SG with ispH and folK knock out E.coli JM109SG with PBPs1b, PBPs5, AmpC and,AmpH ddlB deleted

Jiang et al this lab

Halomonas TD01

Halomonas TD wild type, isolated from a salt lake

27-29

E.coli Rsq01

E.coli BL21(DE3) transformed with plasmid pRsq01-WT

This study

E.coli Rsq02

E.coli BL21(DE3) transformed with plasmid pRsq02-CO

This study

E.coli LTT01 E.coli LTT02 E.coli LTT03 E.coli LTT05 E.coli LTT06 E.coli LTT07

E.coli LTT09

E.coli LTT10 E.coli LTT16 E.coli LTT19 PHB+ ALA+ E.coli LTT19 PHB- ALA+ E.coli LTT19 PHB+ ALA-

E.coli

BL21(DE3)

transformed

with

plasmid

E.coli BL21(DE3) transformed with pLTT02-COH1-K (codon optimized hem1)

plasmid

pLTT01-WTH1 (wild type hem1)

E.coli Origami B (DE3) transformed with plasmid pLTT03- COH1-A E.coli BL21 transformed with plasmid pLTT05H1T7P-C(co-expression with T7 RNA polymerase ) E.coli BL21(DE3) transformed with plasmid pLTT06-H1RM (RhtA& mEos fusion protein) E.coli BL21(DE3) transformed with plasmid pLTT07-H1RLA (HEM1-linker- RhtA ) E.coli JM109SGIK transformed with plasmid pLTT09-H1HKT7P (hem1,ispH, folK co-expression wih T7 RNA polymerase) E.coli JM109SG5△(cell wall weaken strain) transformed with plasmid pQGQ01-H1T7P-K E.coli BL21(DE3) transformed with plasmid pLTT02-COH1 and pVgb E.coli BL21 transformed with plasmid pQGQ01H1T7P-K and p15apCAB E.coli BL21 transformed with plasmid pQGQ01H1T7P-K and p15a E.coli BL21 transformed with plasmid pET28a and p15apCAB

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

This study

This study This study This study This study This study

(To be continued)

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Table I (continued) Strains or Plasmids

Page 30 of 41

Bacterial strains and plasmids used in this study Description

Reference/source

Halomonas TD LTT21

Halomonas TD01 transformed with plasmid pTDALA01

This study

Halomonas TD LTT22

Halomonas TD01 transformed with plasmid pTDALA02

This study

p15a

p15a replicon plasmid

43

p15apCAB

Plasmid expressing phaCAB, p15a-rep ,CmR

43

pET28a

Bacterial expression vector with T7 lac promoter, KanR

Addgene, Inc

pEASY-E2

Bacterial expression vector with T7 lac promoter, AmpR

TransGen Co., Ltd.

pLTT01-WTH1

pET28a inserted with wild type hem1

This study

pLTT02-COH1-K

pET28a inserted with codon optimized hem1

This study

Strains

Plasmids

pRsq01-WT pRsq02-CO

pET28a inserted with wild type Rhodobacter sphaeroides hemA gene pET28a inserted with codon optimized Rhodobacter sphaeroides hemA gene

Biotech

This study This study

pLTT03- COH1-A

pEASY-E2 inserted with codon optimized hem1

This study

pQGQ01- H1T7P-K

pET28a inserted with T7 RNAP and codon optimized hem1, KanR

This study

pLTT05- H1T7P-C

pET28a inserted with T7 RNAP and codon optimized hem1 ( KanR replaced by CmR)

This study

pLTT06-H1RM

pEASY-E2 vector expressing HEM1 and RhtA&mEos fusion protein

This study

pLTT07-H1RLA

pEASY-E2 vector co-expressing connected by a linker

This study

pLTT09-H1HKT7P

pET28a inserted with T7 RNAP, ispH, folK and codon optimized hem1 ( KanR replaced by CmR)

This study

pVgb

pBBR1-MCS1 inserted with vgb, expressing VHb protein

Zhao et al this lab

pRE112-pMB1

pSEVA341-Pporin

HEM1

and

RhtA

pRE112 derivate with pMB1 replication origin, an expression vector in Halomonas TD strain, CmR pSEVA341( pRO1600/ColE1 replication origin,oriT, an expression vector in Halomonas TD strain, CmR) derivate, Porin promoter

26

27

pTDALA01

pRE112-pMB1 inserted with hem1

This study

pTDALA02

pSEVA341-Pporin inserted with hem1

This study

Maps of the plasmids constructed in this study were shown in Supplementary Fig. S1.

30


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Figure 1

Figure 1. Synthetic pathways for ALA and PHB productions constructed in E. coli. Native

C5

pathway

(middle)

synthesizes

ALA from

glutamate

1-semialdehyde

aminotransferase (GSA). The synthetic C4 pathway introduced from Saccharomyces cerevisiae, involves condensation of succinyl-CoA and glycine.(right). Heme, a downstream metabolite of ALA, is a feedback repressor of ALA synthase. T7 RNA polymerase is employed to further strengthen the heterologous expression. To coproduce ALA and PHB, genes phaCAB are added to the recombinant. The substrates for both ALA and PHB production are underlined in the pathways. ALA, 5-aminolevulinic acid; Genes and their enzymes: phaA, β-ketothiolase; phaB: acetoacetyl-CoA reductase; phaC: PHA synthase; hem1, Saccharomyces cerevisiae 5-aminolevulinate synthase encoding gene; ALAS, 5aminolevulinate synthase.

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Page 32 of 41

Figure 2

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Page 33 of 41

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Figure 2. Constructions and screening for suitable strains used for production of ALA and PHB.

(1) Efficient ALA synthase

(ALAS) gene screening, the ALA synthetic capacity of both prokaryotic and eukaryotic ALAS were compared in recombinant E. coli: wild type hemA from Rhodobacter sphaeroides, codon optimized hemA from R. sphaeroides, wild type hem1 from Saccharomyces cerevisiae, codon optimized hem1 from S. cerevisiae;

(2) Screening for high yield recombinants: ALA synthetic abilities of six different

candidate recombinant E. coli including LTT03, LTT10, LTT05, LTT06, LTT06, LTT07 and LTT16 were studied; (3)

Screening for

most suitable E. coli host, E. coli BL21 was found most suitable for ALA production; (4) IPTG-free (auto-induction ) system optimization and antibiotic-free system construction were both carried out based on the most suitable E. coli LTT05; (5) Co-production of ALA and PHB

by recombinant E. coli and Halomonas TD, engineered strains E. coli LTT19 PHB+ALA+ and Halomonas TD

LTT22 were showed.“√” marks the gene or strain selected most suitable for ALA production compared to other candidates, they were used for further engineering improvements .

33



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Page 34 of 41

Figure 3

Figure 3. ALA production by various recombinant E. coli expressing ALA synthase gene. (a) Screening of efficient ALAS gene, strains Rsq01, Rsq02, LTT01 and LTT02 expressed wild type and codon optimized hemA (prokaryotic ALAS, from Rhodobacter sphaeroides), wild

34


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type and codon optimized hem1 (eukaryotic ALAS, from Saccharomyces cerevisiae), respectively. (b) Enhanced ALA production by expressing the codon optimized hem1 in various recombinant E. coli strains: Strain LTT03 expressed codon optimized hem1 in the Origami B(DE3) with its trxB and gor mutated, facilitating the disulfide bonds formation of recombinant protein;

Strain LTT10 expressed codon optimized hem1 in a cell wall weakened

E. coli JM109 with five cell wall synthesis relating genes deleted, allowing easy ALA leakage out of the cells; Strain LTT05 expressed codon optimized hem1 and T7 RNA polymerase gene on plasmid under T7 promoter, further strengthening the T7 system; Strain LTT06 and LTT07 expressed codon optimized hem1 and ALA exporter RhtA. LTT06 strain expressed RhtA as a protein fused with the fluorescent mEos, LTT07 expressed RhtA linked with the ALAS; Strain LTT16 expressed codon optimized hem1 and Vitreoscilla hemoglobin (VHb) encoded by vgb. (c) Comparison of different host strains regarding their ALA production efficiency harboring the same pLTT05- H1T7P-C plasmid as LTT05.

IPTG was added 4 h after incubation with

culture temperature adjusted to 30°C. The pH was stably maintained using phosphate buffer consisting of (mM) 83.4 K2HPO4·3H2O and 216.6 KH2PO4. Each data was obtained from four parallel shake flask cultures.

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Figure 4

Figure 4. ALA production by E. coli LTT09 containing T7 enhanced and antibiotic-free system. (a) Strain LTT09 (right) deleted with essential genes folK and ispH on the genome was able to stably maintained the plasmid expressing folk, ispH, T7 RNA polymerase and hem1 under antibiotic-free conditions, the plasmid was constructed based on the antibiotic stabilized T7 enhanced strain LTT05 (left) containing Cm gene

(b) Growth and ALA production by T7

enhanced and antibiotic-stabilized recombinant E coli LTT05 compared with antibiotic-free LTT09 inoculated in shake flasks. IPTG was added 4 h after incubation with culture temperature adjusted to 30°C. The pH was stably maintained using phosphate buffer consisting of (mM) 83.4 K2HPO4·3H2O and 216.6 KH2PO4. Each data was obtained from four parallel shake flask cultures.

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Figure 5

Figure 5.

Enhanced ALA production by E. coli LTT05 expressing ALA synthase (hem1)

from Saccharomyces cerevisiae under an optimized auto-induction (IPTG-free) system. (a) Buffer optimization (b) Comparison of auto-induction system and IPTG induction time in LB or TB medium (c) Temperature optimization for the auto-induction system. Each data was obtained from four parallel shake flask cultures.

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Figure 6

Figure 6. Co-production of ALA and PHB by recombinant E. coli. (a) Co-production of ALA and PHB allows simultaneous treatments of biomass and supernatant. (b) PHB or/and ALA production by recombinant E. coli. Strain LTT19 PHB+ALA+ expressed hem1, T7 RNA

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polymerase and PHB synthesis operon phaCAB on two plasmids. All recombinant E. coli strains were grown in 100 mL adjusted LB medium with 3-fold phosphate buffers. 3.0 g/L glycine, 6.0 g/L succinic acid were added initially, 4 h later, IPTG and 20 g/L glucose were added and culture temperature was adjusted to 30°C. Additional 10.0 g/L glucose, 5.0 g/L glycine, 10.0 g/L succinic acid were fed at 18 h. The cells were harvested after 48 h.

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Figure 7

Figure 7. Co-production of ALA and PHB by recombinant Halomonas TD.

Halomonas

TD Strain LTT21 harbored pTDALA01 plasmid (pRE112-pMB1 inserted with hem1), Strain LTT22 harbored pTDALA02 plasmid (pSEVA341-Pporin inserted with hem1 under porin promoter). All Halomonas TD strains were grown in 100 mL LB medium containing (g/L) 30 NaCl. 1.0 glycine and 2.0 succinic acid were added at the beginning, culture temperature was adjusted 8 h later to 30°C. Additional (g/L) 10.0 glucose, 2.0 glycine, 4.0 succinic acid fed to the cultures 18 h later. The cells were harvested after 48 h.

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For Table of Contents Use Only

Title: Microbial Synthesis of 5-Aminolevulinic Acid (ALA) and Its Coproduction with Polyhydroxybutyrate (PHB)

Authors: Tian Li, Ying-Ying Guo, Guan-Qing Qiao, Guo-Qiang Chen*


Microbial Synthesis of 5-Aminolevulinic Acid and ... - ACS Publications

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