Metabolic engineering of the terpenoid biosynthetic pathway of ...

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transformation of idi with either dxs or dxr had an additive effect on ß-carotene and zeaxanthin production which reached 1.6 mg g. −1 dry wt. Introduction.
Biotechnology Letters 21: 791–795, 1999. © 1999 Kluwer Academic Publishers. Printed in the Netherlands.

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Metabolic engineering of the terpenoid biosynthetic pathway of Escherichia coli for production of the carotenoids β-carotene and zeaxanthin Manuela Albrecht1 , Norihiko Misawa2 & Gerhard Sandmann1,∗ 1 Biosynthesis

Group, Botanical Institute, Goethe University, P.O. Box 111932, D-60054 Frankfurt, Germany Laboratories for Key Technology, Kirin Brewery, Yokohama, Japan ∗ Author for correspondence (Fax: +49 69 7987 24822; E-mail: [email protected]) 2 Central

Received 17 June 1999; Revisions requested 30 June 1999; Revisions received 19 July 1999; Accepted 20 July 1999

Key words: β-carotene, carotenoid production, engineered E. coli, terpenoid biosynthesis, zeaxanthin

Abstract Metabolic engineering of the early non-mevalonate terpenoid pathway of Escherichia coli was carried out to increase the supply of prenyl pyrophosphates as precursor for carotenoid production. Transformation with the genes dxs for over-expression of 1-deoxy-D-xylulose 5-phosphate synthase, dxr for 1-deoxy-D-xylulose 5-phosphate reductoisomerase and idi encoding an isopentenyl pyrophosphate stimulated carotenogenesis up to 3.5-fold. Cotransformation of idi with either dxs or dxr had an additive effect on ß-carotene and zeaxanthin production which reached 1.6 mg g−1 dry wt.

Introduction Carotenoids are antioxidants with pharmaceutical potential. More than 600 carotenoid structures are known but their availability is limited. Only a few are chemically synthesized and extraction from microorganisms and plant tissue is possible for only a small number of compounds (Johnson & Schroeder 1995). To overcome this problem, heterologous expression of carotenoid genes in Escherichia coli for production of specific carotenoids is an alternative. This approach is suitable for the synthesis of rare carotenoids or even for those which have are not found in Nature. Thus, novel carotenoids can be obtained by combining carotenoid genes from different host species in transgenic E. coli (Albrecht et al. 1997). Several rate-limiting steps for the synthesis and interconversion of carotenoids in E. coli have been elucidated (Ruther et al. 1997, Neudert et al. 1998). However, the amounts of total carotenoids produced in this noncarotenogenic host is limited by the availability of terpenoid precursors which are diverted from biosynthetic pathways of other terpenoids. Therefore, it is

important to engineer the supply of prenyl pyrophosphate precursors for increased carotenoid production. Rate-limitations for the synthesis of carotenoids in E. coli are at the level of prenyl pyrophosphate conversion (Kajiwara et al. 1997, Wang et al. 1999), but little is known about the reaction sequence leading to the synthesis of isopentenyl pyrophosphate (IPP). Like in many other bacteria, IPP is synthesized in E. coli via a novel mevalonate-independent pathway (Rohmer et al. 1996). Only the first two steps, the synthesis of 1deoxy-D-xylulose 5-phosphate (DXP) from pyruvate and glyceraldehyde 3-phosphate and the conversion to 2-C-methyl-D-erythritol 4-phosphate (MEP) are well established. The genes for the enzymes involved, the1-deoxy-D-xylulose 5-phosphate synthase and the reductase been cloned in recent years (Sprenger et al. 1997, Takahashi et al. 1998). These genes will be utilized together with those involved in prenyl pyrophosphate interconversion in this study to remove metabolic bottlenecks by over-production of the appropriate enzymes leading to increased carotenoid production.

792 Table 1. Plasmids used for transformation of E. coli JM101. Plasmid

Replicon

Resistance

Genes

Carotenoids formed

pACCAR161crtXa

p15A

chloramphenicol

β-Carotene

pACCAR251crtXa

p15A

chloramphenicol

pUCBM20dxsb pQEdxrc pMonT-GGSC5 pRK-idi pBBRK-IGC5 pBBRK-dxs

pMB1 pMB1 pMB1 RK2 SC101d SC101d

ampicillin ampicillin ampicillin tetracycline kanamycin kanamycin

crtE, crtB, crtI, crtY, crtE, crtB, crtI, crtY, crtZ dxs dxr crtEC5 idi idi, crtEC5 dxs

Zeaxanthin none none none none none none

a Misawa et al. (1995). b Sprenger et al. (1997). c Takahashi et al. (1998). d Incompatible with SC101.

Materials and methods

Results and discussion

E. coli JM101 transformed with the plasmids listed in Table 1 was grown in LB medium with the appropriate antibiotics in the presence of 0.1 mM IPTG according to Sambrook et al. (1989). After 2 days of growth, cells were harvested and freeze-dried. Carotenoids were extracted from this material with acetone by heating for 15 min at 50 ◦ C and partitioned into 10% ether in petrol and quantified by HPLC on a Nucleosil C18 3 µm column with acetonitrile/methanol/2propanol (85:10:5, by vol.) at 1 ml min−1 . The absorbance spectra of the eluants were recorded on-line with a Water 440 photodiode array detector (Albrecht et al. 1997). Plasmid pRK-idi was constructed by insertion of the reading frame of IPP isomerase gene idi from Xanthophyllomyces dendrorhous (formerly Phaffia rhodozyma) (Accession No. AB019035) into the PstI/BamHI site of pRK404 and pMonT-GGSC5 by PCR amplification of the coding region of the GGPP synthase gene from Sulfolobus acidocaldarius (Ohnuma et al. 1994) and ligation into the XcmI site of a pBluescript derived vector (Borovkov & Rivkin 1997). Plasmid pBBRK-IGC5 was obtained by ligation of both the S. acidocaldarius crtEC5 coding region and the reading frame of the idi gene from X. dendrorhous into the BamHI/XbaI site of vector pBBR1MCS2. Plasmid pBBRK-dxs resulted from the ligation of the coding region of the DXP synthase gene dxs (Sprenger et al. 1997) into the EcoRI/XbaI sites of pBBR1MCS2. The mentioned cloning vectors are described in Table 1.

Carotenoid biosynthesis in E. coli transformed with a carotenogenic gene cluster relies on the supply of geranylgeranyl pyrophosphate (GGPP) which is provided by the endogenous farnesyl pyrophosphate (FPP) synthase in combination with the GGPP synthase of the cluster. The latter is encoded by the crtE gene which is part of plasmids pACCAR161crtX for β-carotene synthesis and pACCAR251crtX for zeaxanthin synthesis (Misawa et al. 1995). Several other plasmids which are described in Table 1 were used for cotransformation with the carotenogenic plasmids mentioned above. They contain on compatible vectors all known genes of the established early enzymatic steps of the non-mevalonate terpenoid pathway leading to IPP synthesis. These are the dxs gene encoding the initial DXP synthase and dxr the subsequent reductoisomerase which were additionally expressed including a foreign IPP isomerase gene, idi from X. dendrorhous. The expression of these three genes could be demonstrated after protein separation on an SDS polyacrylamide gel as new protein bands of correct molecular weights (data not shown). A special GGPP synthase gene from S. acidocaldarius encodes an enyzme which by-passes FPP synthesis by direct conversion of the substrate DMAPP to GGPP covering the enitre chain elongations to C20 (Ohnuma et al. 1994) was also used to enhance carotenogenesis. Expression of this gene in E. coli was confirmed by complementation of carotenoid synthesis of a carotenogenic gene cluster devoid of a GGPP synthase gene (data not shown).

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Fig. 1. Terpenoid pathway in E. coli and formation of carotenoids in transformants with a carotenogenic gene cluster. Foreign or additionally expressed genes are indicated at the corresponding reaction. Symbol ⊕ indicates stimmulation of carotenoid synthesis. DMAPP = dimethylallyl pyrophsphate, IPP = isopentenyl pyrophosphate, FPP = farnesyl pyrophosphate, GGPP = geranylgeranyl pyrophosphate.

All the reactions mentioned and the genes involved are outlined in Figure 1. Due to the presence of plasmids pACCAR161crtX and pACCAR251crtX the carotenoids ß-carotene and zeaxanthin were accumulated to a concentration close to 500 µg mg−1 dry weight in E. coli JM101 with nonmodified early terpenoid synthesis (Table 2). These concentrations of β-carotene and zeaxanthin were doubled upon co-transformation of E. coli with either the plasmid pUCBM20dxs containing the dxs or pRKidi with the idi gene. When the dxs and idi-containing plasmids were introduced simultaneously, the effect was additive resulting in 1.5 mg of β-carotene or zeaxanthin g−1 dry wt. After co-transformantion with the dxr gene also higher zeaxanthin and β-carotene contents were observed. The presence of this second gene of the early terpenoid pathway resulted in an increase of about 50%. As observed for dxs, a combination of dxr with idi led to an additive effect on carotenoid synthesis and an up to a 2.4-fold higher accumulation compared to a carotenogenic transformant with an non-modified pathway to DMAPP. On plasmid pMonT-GGSC5, a gene for a GGPP synthase is introduced which covers also the step to FPP by using DMAPP as allylic substrate (Figure 1).

An increase of the synthesis of β-carotene and zeaxanthin was not observed in its presence (Table 2). In combination with idi on pBBRK-IGC5 and an additional dxs gene even a slight negative effect was found. As summarized in Figure 1, over-expression of three enzymes of the E. coli terpenoid pathway, 1deoxy-D-xylulose 5-phosphate synthase, 1-deoxy-Dxylulose 5-phosphate reductoisomerase and IPP isomerase led to a stimulation of the synthesis of either β-carotene or zeaxanthin. In a report on the effect of a DMAPP converting archebacterial GGPP synthase on formation of the carotenoid astaxanthin (Wang et al. 1999), this type of GGPP synthase produced a similar effect on carotenoid synthesis as the crtE encoded GGPP synthase from Erwinia. In combination with idi, a strong increase compared to the crtE/idi combination was observed. However, this finding could not be confirmed in our investigation using the gene of a biochemically equivalent enzyme from S. acidocaldarius (Table 2). The positive effect of idi overexpression alone on carotenoid synthesis reported by these authors and also found by us has been shown before (Kajiwara et al. 1997).

794 Table 2. Formation of β-carotene and zeaxanthin upon egineering of the terpenoid metabolism in E. coli. Plasmid combination

Carotenoid µg g−1 dry wt (%)

β-Carotene pACCAR161crtX 440 (100) + pUCBM20dxs 920 (209) + pQEdxr 704 (160) + pRK-idi 928 (211) + pUCBM20dxs + pRK-idi 1533 (348) + pQEdxr + pRK-idi 1066 (242) + pMonT-GGSC5 395 (90) + pBBRK-dxs + pMonT-GGSC5 805 (183) + pBBRK-IGC5 712 (162) + pUCBM20dxs + pBBRK-IGC5 1368 (311) pACCAR251crtX + pUCdxs + pQEdxr + pRK-idi + pUC BM20dxs + pRK-idi + pQEdxr + pRK-idi + pMonT-GGSC5 + pBBRK-dxs + pMonT-GGSC5 + pBBRK-IGC5 + pUCBM20dxs + pBBRK-IGC5

Zeaxanthin 495 (100) 977 (197) 722 (146) 1094 (221) 1570 (317) 1118 (226) 545 (110) 904 (183) 811 (164) 1451 (293)

Carotenoid values are means of at least 3 determinations; all standard deviations were in the range of ±15% of the means.

Looking at the interaction of the individual reactions of terpenoid biosynthesis in E. coli, it is evident that not only the initial enzyme of the early terpenoid pathway to prenyl pyrophosphates is able to push more metabolites into the biosynthetic chain, but also the dxr product can do so by pulling its substrate 1-deoxyD-xylulose 5-phosphate. Furthermore, when the limitation at the level of IPP isomerisation is overcome by expression of higher enzyme levels, an additive effect with overproduction of one of the initial enzymes was revealed. All these results indicate that the terpenoid pathway of E. coli is not regulated by a single ratelimiting step. In contrast, an increase of most of the known enzymes has an overall effect on the capacity of the pathway. We tried to investigate the combination effect of the dxs and dxr genes on carotenogenesis. For this purpose competent cells of E. coli JM101 containing either plasmid pACCAR161crtX or pBBRK-dxs were co-transformed with two additional compatible plasmids with different antibiotic resistances (Table 1), pBBRK-dxs + pQEdxr and pACCAR161crtX +

pQEdxr, respectively (Table 3). In none of these cases the transformant JM101/pACCAR161crtX/pBBRKdxs/pQEdxr containing all three plasmids was obtained nor after stepwise transformations with one plasmid at a time. However, plating on selected mixtures of antibiotics revealed that two of the mentioned plasmids in all combinations could be introduced into E. coli. Thus, we obtained transformants which contained either both genes of the early pathway, dxs and dxr, or each of these genes together with plasmid pACCAR161crtX which is responsible for carotenoid synthesis. Therefore, it can be concluded that a transformant with all three genes seems to be lethal (Table 3). The same was also found in similar transformations when pACCAR161crtX was replaced by pACCAR251crtX (data not shown). This result is interpreted in the following way: It is assumed that the increase of the activities of the enzymes DXS and DXR which leads to an accelerated formation of prenyl pyrophosphates as indicated by a subsequent higher synthesis of carotenoids (Table 2) are additive. The very high activities of IPP synthesis and high yields of prenyl pyrophosphates obviously do not cause a general problem for the metabolism of E. coli, as transformant JM101/pBBRK-dxs/pQEdxr was obtained easily (Table 3) and no impairment of growth was observed. Thus, the formation and accumulation of carotenoids expected in concentrations somewhere beyond the highest value of 1570 µg g−1 dry wt in Table 2 when pACCAR161crtX is additionally present creates the problem for E. coli. A carotenogenic host should possess a high storage capacity for carotenoids (Sandmann et al. 1999) otherwise, the lipophilic carotenoids will overload the membranes and block their functionality. Obviously, this limit must have been reached by overexpression of DXS and DXR simultaneously with a carotenogenic gene cluster. Therefore, the future focus for development of transgenic E. coli as a production system for carotenoids should be on the formation of additional membranes in E. coli which can be achieved by transformation of a special gene (Weiner et al. 1984) and on other genetic manipulations leading to novel carotenoid sequestering systems, e.g., soluble carotenoid binding proteins within the cells.

795 Table 3. Multiple transformation of E. coli JM101 with pACCAR161crtX, pBBRK-dxs and pQEdxr. Competent cells and applied plasmids

Growth of transformants on combinations of antibioticsa (additionally introduced plasmid)

A. JM101/pACCAR161crtX + pBBRK-dxs + pQEdxr

chl + kan + amp

chl + kan

chl + amp

negativeb

positivec (pBBRK-dxs)

positivec (pQEdxr)

kan + chl + amp

kan + chl

kan + amp

negatived

positivec (pACCAR161crtX)

positive (pQEdxr)

B. JM101/pBBRK-dxs + pACCAR161crtX + pQEdxr

a kan = kanamycin, chl = chloramphenicol, amp = ampicillin. b Control tranformation with pBBR1MCS2 together with pQE30 was successful. c Resulting transformants were pigmented. d Control tranformation with pACCAR161crtX together with pQE30 was successful.

Acknowledgements Our due thanks are expressed to Dr G. Sprenger and Sigrid Grolle, Jülich, Germany for provision and construction of plasmids pUCBM20dxs and pBBRK-dxs. We are grateful to Dr A. Brokov, Monsanto, St. Louis, USA for the pBluescript derived T-overhang vector and to Dr C. Schleper, Technische Universität, Darmstadt, Germany and Dr T. Nishino, Tohoku University, Japan for supplying us with DNA of Sulfolobus acidocaldarius.

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