Biosynthesis of cycloartenol by expression of plant and bacterial oxidosqualene cyclases in engineered Rhodobacter capsulatus

Journal Pre-proof Biosynthesis of cycloartenol by expression of plant and bacterial oxidosqualene cyclases in engineered Rhodobacter capsulatus Jennifer Hage-Hulsmann ¨ (Investigation) (Formal analysis) (Visualization) (Writing - original draft) (Writing - review and editing), Sabine Metzger (Formal analysis) (Validation) (Writing - review and editing), Vera Wewer (Investigation) (Formal analysis) (Visualization) (Writing - review and editing), Felix Buechel (Investigation) (Writing review and editing), Katrin Troost (Investigation) (Writing - review and editing), Stephan Thies (Validation) (Writing - review and editing), Anita Loeschcke (Supervision) (Validation) (Writing - review and editing), Karl-Erich Jaeger (Conceptualization) (Funding acquisition) (Writing - review and editing), Thomas Drepper (Conceptualization) (Validation) (Writing - review and editing)

PII:

S2590-1559(20)30001-9

DOI:

https://doi.org/10.1016/j.btecx.2020.100014

Reference:

BTECX 100014

To appear in:

Journal of Biotechnology: X

Received Date:

15 August 2019

Revised Date:

14 January 2020

Accepted Date:

29 January 2020

Please cite this article as: Hage-Hulsmann ¨ J, Metzger S, Wewer V, Buechel F, Troost K, Thies S, Loeschcke A, Jaeger K-Erich, Drepper T, Biosynthesis of cycloartenol by expression of plant and bacterial oxidosqualene cyclases in engineered Rhodobacter capsulatus, Journal of Biotechnology: X (2020), doi: https://doi.org/10.1016/j.btecx.2020.100014

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Biosynthesis of cycloartenol by expression of plant and bacterial oxidosqualene cyclases in engineered Rhodobacter capsulatus

Jennifer Hage-Hülsmanna,b, Sabine Metzgerb,c, Vera Wewerb,c, Felix Buechelb,c, Katrin Troosta,d, Stephan Thiesa,d, Anita Loeschckea,b,d, Karl-Erich Jaegera,b,d,e, Thomas Dreppera,b

Institute of Molecular Enzyme Technology, Heinrich Heine University Düsseldorf,

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Forschungszentrum Jülich, Jülich, D-52425, Germany

Cluster of Excellence on Plant Sciences (CEPLAS) Düsseldorf, D-40225, Germany

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MS Platform, Department of Biology, University of Cologne, Cologne, D-50674 Germany

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Bioeconomy Science Center (BioSC), Forschungszentrum Jülich, Jülich, D-52425, Germany

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Institute of Bio- and Geosciences IBG-1, Forschungszentrum Jülich, Jülich, D-52425, Germany

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b

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E-mail addresses of all authors:

Jennifer Hage-Hülsmann, [email protected] Sabine Metzger, [email protected]

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Vera Wewer, [email protected]

Felix Buechel, [email protected] Katrin Troost, [email protected]

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Stephan Thies, [email protected]

Anita Loeschcke, [email protected] Jaeger, Karl-Erich, [email protected]

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Thomas Drepper, [email protected]

Corresponding authors:

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Anita Loeschcke, [email protected] Thomas Drepper, [email protected]

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Highlights

R. capsulatus is suitable for expression of plant/microbial cycloartenol synthases

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Titers were independent of chosen plant and myxobacterial cycloartenol synthases

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Co-expression of the mevalonate pathway had little impact on cycloartenol levels

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Deletion of crtE led to threefold increased cycloartenol product titers

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Abstract

Cyclic triterpenes are a large group of secondary metabolites produced by plants, fungi and bacteria. They have diverse biological functions, and offer potential health benefits for humans. Although various

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terpenes from the mono-, sesqui- and diterpene classes are easy to produce in engineered bacteria, heterologous synthesis of cyclic triterpenes is more challenging. We have recently shown that the

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triterpene cycloartenol can be produced in Rhodobacter capsulatus SB1003 but initial titers were low with 0.34 mg L-1. To assess, if this phototrophic α-proteobacterium can be engineered for enhanced

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triterpene production, we followed two alternative strategies by comparing the performance of the R. capsulatus SB1003 wildtype strain with two recombinant strains carrying either a mevalonate pathway implemented from Paracoccus zeaxanthinifaciens or a deletion in the intrinsic carotenoid

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biosynthesis gene crtE. These strains are thus engineered for an enhanced isoprenoid biosynthesis or a suppressed precursor conversion by the competing carotenoid pathway. Moreover, three different

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cycloartenol synthase (CAS) genes from Arabidopsis thaliana or the myxobacterial strains Stigmatella aurantiaca Sg a15 and DW4/3-1 were tested for heterologous cycloartenol synthesis. We found that the

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heterologous expression of mevalonate pathway enzymes had little impact on cycloartenol levels irrespective of the chosen CAS. In contrast, the use of the newly constructed carotenoid-deficient crtE deletion strain showed threefold increased cycloartenol product titers. We conclude that R. capsulatus is a promising alternative host for the functional expression of triterpene biosynthetic enzymes from plants and microbes. Apparently, product titers can also be improved by suppression of competing precursor consumption.

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Key words: cyclic triterpene biosynthesis, heterologous metabolic pathway, cycloartenol synthase, metabolic engineering, OSC, Rhodobacter

1. Introduction Terpenes constitute one of the largest groups of secondary metabolites and are found in all kingdoms of life. Plants produce several thousand diverse terpene structures, including phytosterols of the triterpene class (Thimmappa et al., 2014). Basically, all terpenes are synthesized from the isoprene

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precursors isopentenyl pyrophosphate (IPP) and dimethylallyl pyrophosphate (DMAPP) via the 2-C-methyl-D-erythritol 4-phosphate (MEP) or the mevalonate (MVA) pathway. IPP and DMAPP are C5 compounds which are consecutively condensed to form the C10 geranyl pyrophosphate (GPP), and

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subsequently the C15 derivative farnesyl pyrophosphate (FPP). For triterpene biosynthesis, two molecules of FPP are condensed by squalene synthase (SQS) to form the C30 product squalene. This

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in turn is epoxidized by a squalene epoxidase (SQE) to 2,3-oxidosqualene, which finally acts as a central precursor to form more specialized triterpene products, catalyzed by a family of enzymes known as

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oxidosqualene cyclases (OSC). This complex catalytic reaction involves a stereoselective cationic cyclization and skeletal rearrangements, typically terminated by deprotonation to yield the final cyclic scaffold. One such enzyme is the cycloartenol synthase (CAS), which converts 2,3-oxidosqualene into

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cycloartenol via substrate folding into the chair-boat-chair (CBC) conformation and generation of a protosteryl cationic intermediate by protonation before a series of hydride and methyl shifts and finally

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deprotonation result in the first dedicated tetracyclic C30 scaffold of the phytosterol pathway (Thimmappa et al., 2014).

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Large quantities of cycloartenol are found in natural plant oils such as olive oil and avocado oil (Mo et al., 2013), and the phytosterol content of these products is associated with the health benefits of these products (Kerrihard and Pegg, 2015). In addition, the anticancer activity of cycloartenol has recently raised interest (Ishola and Adewole, 2019; Niu et al., 2018). The biosynthesis of this compound is generally attributed to plantal phytosterol synthesis, but interestingly also occurs in fungi and even some prokaryotes, including the myxobacterium Stigmatella aurantiaca, where their function remains to be elucidated (Bode et al., 2003; Desmond and Gribaldo, 2009; Wei et al., 2016).

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Since a strong growth of the phytosterol market is anticipated but the extraction from plants can be associated with difficulties regarding sustainability, reliability and purity, biotechnological synthesis of cyclic triterpenes in microbes may pose an attractive alternative to access these compounds (Kallscheuer et al., 2019). Prokaryotes have been extensively used for the heterologous production of several classes of terpenes (Khan at al., 2015; Li and Wang, 2016; Pitera et al., 2007; Schempp et al., 2018; Wu et al., 2017), but the functional expression of plant OSCs in bacteria appears to remain a major challenge and reports on the synthesis of cyclic triterpenes in bacteria are scarce (Moser and Pichler, 2019). We have previously shown that plant triterpene biosynthesis pathways can, in principle, be incorporated into the metabolically versatile photosynthetic α-proteobacterium Rhodobacter

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capsulatus SB1003 (Loeschcke et al., 2017). This carotenogenic bacterium synthesizes IPP and DMAPP via the MEP pathway, converts them into FPP and subsequently into the C20 compound geranylgeranyl pyrophosphate (GGPP) via the enzyme GGPP synthase encoded by the crtE gene.

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GGPP is the direct precursor of C40 tetraterpenes, and is used in R. capsulatus to produce the carotenoids spheroidene and spheroidenone (Armstrong, 1997) (Fig. 1A).

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We showed in a previous study that the intrinsic metabolic capability of the bacterium can be utilized to produce plant-identical cyclic triterpenes via concerted heterologous expression of Arabidopsis thaliana

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squalene synthase 1 (SQS1), squalene epoxidase 1 (SQE1) and an oxidosqualene cyclase (OSC) but achieved limited triterpene titers (e.g. 0.34 mg L-1 in the case of cycloartenol) in the respective wildtype background (Loeschcke et al., 2017). We have further demonstrated that production of

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sesquiterpenoids, which also derive from FPP, can be enhanced by introduction of the MVA pathway [R. capsulatus SB1003-MVA, (Troost et al., 2019)]. Alternatively, the production of isoprenoids may be

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enhanced by decreasing FPP consumption in the intrinsic tetraterpene biosynthetic pathway via deletion of GGPP synthase-encoding crtE (Fig. 1A). In addition, it was demonstrated that the type and source

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of heterologous biocatalysts used for implementation of recombinant terpenoid biosynthesis represent key determinants affecting product levels (Qiao et al., 2019; Troost et al., 2019). Here, we analyzed cycloartenol production (i) in R. capsulatus strains SB1003, SB1003-MVA and newly constructed SB1003-ΔcrtE, as well as (ii) by comparative expression of CAS-encoding genes from A. thaliana and the myxobacterial strains S. aurantiaca Sg a15 and DW4/3-1.

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2. Results and discussion We previously showed that cyclic triterpenes from plants can be produced in the α-proteobacterium R. capsulatus SB1003 by expression of biosynthetic genes from A. thaliana, although the product titers of cycloartenol were low (Loeschcke et al., 2017). In an attempt to increase the titers of this phytosterol, we now compared the suitability of different R. capsulatus strains for functional expression of different CAS enzymes. Besides the wildtype strain SB1003, we employed strain SB1003-MVA (Troost et al., 2019), which carries the MVA pathway gene cluster from the bacterium P. zeaxanthinifaciens that provides an additional route for the conversion of acetyl-CoA into C5 isoprene units. In addition, we

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constructed strain SB1003-ΔcrtE by interposon mutagenesis to block FPP consumption for carotenoid biosynthesis via GGPP synthase CrtE, which should therefore increase the availability of FPP for the heterologous pathway (Fig. 1A). All three strains were used for Pnif promoter-based expression of codon usage adapted triterpene biosynthetic genes using the pRhon5Hi-2 expression vector (Fig. 1B) (Troost

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et al., 2019). Strain engineering (Fig. S1, Table S1) and vector construction (Fig. S2, Table S2), microaerobic cultivation, cell extraction and LC-MS analysis of terpenoids are described in the

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Supplementary methods.

Initially, the gene encoding A. thaliana squalene synthase 1 (SQS1) was expressed alone and together

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with the gene encoding A. thaliana squalene epoxidase 1 (SQE1) in the three strains to confirm the ability of the bacteria to accumulate the precursors squalene and 2,3-oxidosqualene. Indeed, LC-MS analysis of cell extracts showed that all strains produced the precursors (Fig. S3). In all strains, squalene

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was largely consumed when SQE1 was co-expressed (data not shown). Remarkably, higher accumulation levels of squalene or 2,3-oxidosqualene could be observed for the R. capsulatus ΔcrtE

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mutant expressing either SQS1 (Fig. S3A) or SQS1 and SQE1 (Fig. S3B) in comparison to the respective wildtype and MVA strains as indicated by an increase of in LC-MS signal intensities. Next,

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we tested the ability of the three strains expressing SQS1 and SQE1 to produce cycloartenol by comparative co-expression of genes encoding CAS enzymes (Fig. 1C). We used the CAS1 of A. thaliana (here referred to as At-CAS) as a reference, and the OSCs from S. aurantiaca Sg a15 and S. aurantiaca DW4/3-1 (here referred to as SaSg-CAS and SaDW-CAS, respectively), which have not been used for heterologous expression and triterpene biosynthesis before. In the wildtype strain SB1003, expression of all tested CAS genes resulted in comparable cycloartenol titers of ca. 350 µg L1

. While for the expression of At-CAS, this result was expected based on our previous study (Loeschcke

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et al., 2017), it was interesting to note that the implementation of these bacterial enzymes, which were used for heterologous expression for the first time, led to the same product levels.

A DMAPP (C5) IspA IspA GPP (C10) Idi IPP (C5)

SQS

MVA pathway

squalene (C30) SQE

GGPP (C20) Carotenoid biosynthesis Crt enzymes

2,3-oxidosqualene (C30) pyruvate + G3P

spheroidene/ spheroidenone

CBC conformation

acetyl-CoA

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R. capsulatus

MEP pathway

CrtE FPP (C15)

CAS

protosteryl cation

intrinsic central metabolism

photosynthesis/ photoprotection

C MVA

SQS1 SQE1 CAS

R. capsulatus SB1003-MVA chromosome

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pRhon5Hi-2

1.0 1000

mg/L

Pnif

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Pnif

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SQS1 from A. thaliana SQE1 from A. thaliana R. capsulatus SB1003-ΔcrtE CAS from: chromosome A. thaliana (At-CAS1), crtE S. aurantiaca Sg a15 (SaSg-CAS), or S. aurantiaca DW4/3-1 (SaDW-CAS)

cycloartenol titer [µg L-1]

B

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cycloartenol (C30)

0.5 500

0 0.0 origin of CAS enzyme

R. capsulatus

SB1003

SB1003-MVA SB1003-ΔcrtE

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Fig. 1. Recombinant phytosterol production in engineered R. capsulatus. (A) Intrinsic and engineered isoprenoid metabolism of R. capsulatus: In the wildtype strain SB1003, the MEP pathway uses the building blocks glyceraldehyde 3-phosphate and pyruvate of the central metabolism to provide isoprene precursors (gray), which are elongated and utilized to biosynthesize carotenoids for photosynthesis and photoprotection (red). The previously established strain SB1003-MVA was equipped with the MVA pathway from the bacterium Paracoccus zeaxanthinifaciens (blue), and thus provides an additional route for the conversion of acetyl-CoA into isoprenoids. In the newly constructed strain SB1003-ΔcrtE, the metabolic branch which consumes FPP for carotenoid synthesis is blocked due to deficiency of GGPP synthase CrtE. Heterologous triterpene synthesis (green) can be implemented by conversion of FPP to squalene, which is further epoxidized to 2,3-oxidosqualene, the precursor of more complex triterpenes, which can in turn be converted to cycloartenol. MEP, 2-C-methylD-erythritol 4-phosphate; G3P, glyceraldehyde 3-phosphate; MVA, mevalonate; IPP, isopentenyl pyrophosphate; DMAPP, dimethylallyl pyrophosphate; GPP, geranyl pyrophosphate; FPP, farnesyl pyrophosphate; GGPP, geranylgeranyl pyrophosphate; Idi, isopentenyl diphosphate isomerase IspA, FPP synthase; CrtE, GGPP synthase; SQS, squalene synthase; SQE, squalene epoxidase; CAS, cycloartenol synthase; CBC, chair-boat-chair. (B) Schematic of employed expression vectors that carry genes encoding SQS1, and SQE1 from A. thaliana, as well as a CAS enzyme either from A. thaliana, or from S. aurantiaca. Vectors were used for Pnif-based gene expression in the R. capsulatus wildtype

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SB1003 and the engineered strains SB1003-MVA and SB1003-ΔcrtE. (C) Cycloartenol titers in R. capsulatus expression cultures [µg L-1], determined by LC-MS analysis of cell extracts upon microaerobic cultivation for 2 days. Values represent means from three independent cultivations and measurements with error bars indicating the respective standard deviation.

Slightly higher cycloartenol levels were achieved when the same CAS genes were expressed in strain SB1003-MVA, with titers of ca. 540 µg L-1. However, CAS gene expression in the new carotenoiddeficient strain SB1003-ΔcrtE resulted in clearly elevated cycloartenol titers of 1130 µg L-1 (At-CAS), 900 µg L-1 (SaSg-CAS) and 840 µg L-1 (SaDW-CAS). Final cell densities were comparable; they are summarized along with respective specific cycloartenol yields per gram dry cell weight (mg gDCW-1) and specific productivities (µg gDCW-1 h-1), together with the reached titers in the supplementary data

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(Table S3, Fig. S4). Although a strong dependency of titers on specific terpene synthase enzymes has been reported oftentimes, e.g. for the heterologous production of valencene in Rhodobacter sphaeroides and R. capsulatus (Beekwilder et al., 2014; Troost et al., 2019), we did not observe this

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effect here.

Interestingly, unconverted 2,3-oxidosqualene was detected in cycloartenol producing strains, especially

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with the enzyme SaSg-CAS (Fig. S5), indicating that the availability of this precursor is not the key limiting factor for the production of cycloartenol. Moreover, an additional signal was observed in LC-MS

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analyses upon the expression of SaSg-CAS (Fig. S6). Based on the detected masses, it might be speculated to be an oxygenated derivative of cycloartenol. The heterologous production of cycloartenol in yeast cells expressing plant-derived CAS has been reported several times, but these studies were

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focused on enzyme functionality rather than metabolic engineering and, to our knowledge, the product titers were not determined (Corey et al., 1993; Jin et al., 2017; Sandeep et al., 2019; Zhang et al., 2003).

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Recently, bacterial hosts such as E. coli, R. capsulatus and Synechocystis sp. have been used for the heterologous expression of OSCs from plants (Li et al., 2016; Loeschcke et al., 2017; Qiao et al., 2019;

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Takemura et al., 2017) resulting in the production of both pentacyclic and sterol-type molecular scaffolds. The use of bacterial OSC enzymes, which are much less prevalent, has only rarely been described (Banta et al., 2017). In E. coli and R. capsulatus, this has so far led to titers in the lower µg range (Loeschcke et al., 2017; Takemura et al., 2017). Here, we were able to improve the productivity of R. capsulatus about threefold by using the engineered strain SB1003-ΔcrtE for expression of SQS1 and SQE1 from A. thaliana, as well as CAS enzymes from A. thaliana or of myxobacterial origin, resulting in cycloartenol titers of up to 1130 µg L-1.

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In a similar R. capsulatus-based approach it was previously reported that the heterologous production of linear triterpenoids botryococcene and squalene via expression of respective synthases from Botryococcus braunii could be significantly enhanced by engineering of the intrinsic isoprenoid precursor biosynthesis from well below 0.5 mg gDCW-1 to ca. 2 and ca. 9 mg gDCW-1 in standard growth medium, respectively. This was achieved via co-expression of an additional FPP synthase and rate-limiting enzymes DxS synthase and IPP isomerase of the MEP pathway (Khan et al., 2015).Here, we employed the engineered strain SB1003-MVA, which carries a heterologous MVA pathway to increase the synthesis of the precursors IPP and DMAPP, which was shown to strongly enhance sesquiterpene product levels in our previous study (Troost et al., 2019). However, in the case of cycloartenol

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production, this standard approach for metabolic engineering of terpenoid pathways only resulted in a marginal increase of final product levels (Fig. 1C). In many studies, metabolic engineering has been used to increase the production of various valuable molecules, but especially the terpenoid pathway is

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controlled by a complex regulatory network and titers can be influenced by factors such as feedback inhibition, e.g. due to the accumulation of intermediates (Dahl et al., 2013; Park et al., 2017; Pitera et

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al., 2007). Our results with the additional MVA pathway might be due to inhibitory effects which limit the productivity of these cells, e.g. at the level of the host’s own FPP synthase, which may be inhibited by

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high concentrations of its substrate IPP, as has been demonstrated for the human enzyme (Barnard and Popják, 1981; Park et al., 2017). Further, an inhibition of the employed squalene synthase from A. thaliana by excess FPP is thinkable, as observed for the respective enzyme from yeast (Zhang et al.,

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1993). In both cases, use of additional or alternative IspA and SQS enzymes with potentially different properties, as explored by Curtis and coworkers (Khan et al., 2015), in combination with the MVA

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pathway might be a promising approach for future research. In contrast, deletion of crtE-encoded GGPP synthase led to increased cycloartenol levels, which may be assigned to different factors including not

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only a decrease in intrinsic FPP consumption and thus favored flux into triterpene synthesis, but potentially also to a loss of feedback inhibition of isoprenoid biosynthetic steps due to the absence of the natural carotenoid end products. Finally, cells of this strain should encounter a significantly lower metabolic burden compared to the wildtype or the strain additionally expressing the MVA pathway enzymes. However, the underlying processes yet need to be uncovered in future studies. Finally, besides metabolic engineering, the optimization of cultivation conditions including the selection of an appropriate carbon source and exploitation of the Rhodobacter physiological capacities can help to further increase final product titers as previously described for the production of squalene in

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R. capsulatus (Khan et al., 2015) and amorphadiene in Rhodobacter sphaeroides (Orsi et al., 2019). Therefore, comprehensive bioprocess engineering approaches are needed to further elevate production levels and to assess if an economically attractive triterpene biosynthesis process can be established using R. capsulatus.

Conclusions: We demonstrate here that the valuable phytosterol cycloartenol can be produced in R. capsulatus using plant and bacterial OSC enzymes, in particular when steps are taken to tune precursor availability: We provide a case study where the deletion of a competing intrinsic metabolic route was more effective than the addition of an alternative route to precursors or the use of alternative

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OSC enzymes.

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Abbreviations MEP, 2-C-methyl-D-erythritol 4-phosphate; MVA, mevalonate; G3P, glyceraldehyde 3-phosphate; IPP, isopentenyl pyrophosphate; DMAPP, dimethylallyl pyrophosphate; GPP, geranyl pyrophosphate; FPP, farnesyl pyrophosphate; GGPP, geranylgeranyl pyrophosphate; Idi, isopentenyl diphosphate isomerase IspA, FPP synthase; CrtE, GGPP synthase; SQS, squalene synthase; SQE, squalene epoxidase; OSC, oxidosqualene cyclase; CAS, cycloartenol synthase; CBC, chair-boat-chair, At-CAS, A. thaliana CAS1; SaSg-CAS, OSC from S. aurantiaca Sg a15; SaDW-CAS, OSC from S. aurantiaca

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DW4/3-1.

Declaration of interests

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The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

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Author Contributions

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Jennifer Hage-Hülsmann: Investigation, Formal analysis, Visualization, Writing – original draft, review & editing Sabine Metzger: Formal analysis, Validation, Writing – review & editing Vera Wewer: Investigation, Formal analysis, Visualization, Writing – review & editing Felix Buechel: Investigation, Writing – review & editing Katrin Troost: Investigation, Writing – review & editing Stephan Thies: Validation, Writing – review & editing Anita Loeschcke: Supervision, Validation, Writing – review & editing Karl-Erich Jaeger: Conceptualization, Funding acquisition, Writing – review & editing Thomas Drepper: Conceptualization, Validation, Writing – review & editing

Acknowledgements We gratefully acknowledge the Deutsche Forschungsgemeinschaft for funding JHH, SM, VW and FB via the Cluster of Excellence on Plant Science (CEPLAS) (EXC1028). KT, ST, and AL were funded by the Ministry of Culture and Science of the German State of North Rhine-Westphalia (through NRW

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Strategieprojekt BioSC (No. 313/323-400-00213). The authors thankfully acknowledge Dr. Richard M. Twyman for providing advice on text-editing.

Supplementary information The Supplementary data file provides detailed information on methodology for strain engineering, vector construction including DNA and respective protein sequences, microaerobic cultivation, cell

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extraction and LC-MS analysis of terpenoids.

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