Dual pathway for metabolic engineering of E. coli metabolism to produce the highly valuable hydroxytyrosol

One of the most abundant phenolic compounds traced in olive tissues is Hydroxytyrosol (HT), a molecule that has been attributed with a pile of beneficial effects, well documented by many epidemiological studies and thus adding value to products containing it. Strong antioxidant capacity and protection from cancer are only some of its exceptional features making it ideal as a potential supplement or preservative to be employed in the nutraceutical, agrochemical, cosmeceutical, and food industry. The HT biosynthetic pathway in plants (e.g. olive fruit tissues) is not well apprehended yet. In this contribution we employed a metabolic engineering strategy by constructing a dual pathway introduced in Escherichia coli and proofing its significant functionality leading it to produce HT. Our primary target was to investigate whether such a metabolic engineering approach could benefit the metabolic flow of tyrosine introduced to the conceived dual pathway, leading to the maximalization of the HT productivity. Various gene combinations derived from plants or bacteria were used to form a newly-inspired, artificial biosynthetic dual pathway managing to redirect the carbon flow towards the production of HT directly from glucose. Various biosynthetic bottlenecks faced due to feaB gene function, resolved through the overexpression of a functional aldehyde reductase. Currently, we have achieved equimolar concentration of HT to tyrosine as precursor when overproduced straight from glucose, reaching the level of 1.76 mM (270.8 mg/L) analyzed by LC-HRMS. This work realizes the existing bottlenecks of the metabolic engineering process that was dependent on the utilized host strain, growth medium as well as to other factors studied in this work.


Introduction
158 The asterisks in aroG* and tyrA* refer to the feedback-resistant variants of aroG and tyrA, respectively. 159 OP designation stands for a tyrosine overproducer plasmid or strain. 173 Genes were either cloned by PCR utilizing appropriately designed oligonucleotides from various 174 sources (Table S1), or artificially synthesized (Table S1). To confirm each gene's identity the cloned 175 PCR fragments were sequenced and compared in silico with the NCBI deposited sequences.
250 The phenylacetaldehyde reductase gene (PAR) from Rhodococcus erythropolis (RePAR) was 251 amplified with the primer pair RePARfw2/RePARrv2 (Table S1). The amplified DNA fragment was 252 digested with the restriction enzymes NcoI and HindIII and was inserted into the respective sites of an 253 empty pET Duet vector resulting the pET-RePAR vector (Table 2) (Table 2).

In-vivo expression and growth optimization experiments
264 All genes referred in Table 1 were used primarily in in-vivo experiments, in order to check their optimal 265 expression as well as their function. The latter was achieved by measuring their activity of their 266 respective enzyme product activity through the quantity of their equivalent end-product biosynthesis.
267 Two growth protocols were used in this work, targeting the maximal production of each intermediate  Table 2). Six 371 of these genes were necessary for the biosynthesis of shikimic acid and five of them required for the 372 conversion of shikimic acid into tyrosine. The shikimic acid biosynthesis module (pS4; Fig S2) Table 2), in induced conditions were checked initially for their ability to induce the expression of the 388 TYR protein (Fig S3a). As negative controls BL21 cells were used transformed either with empty 389 pRSF vector in non-induced conditions or with BL21-pRSF-RsTYR in non-induced conditions. It was 390 obvious that a band with much higher intensity (see arrow in Fig S3a)  437 Similarly to TYR in-vivo assays, the cultures under non-induced conditions presented minimal 438 consumption of the substrate (Fig 5 A and C) while the induced conditions exerted the maximal 439 conversion efficiency (Fig 5 B and D).  (Table 1, Table 2, Table S1)  Hydroxytyrosol production from engineered tyrosine-overproducer E. coli 506 strains 507 Instead of using an E. coli system that is necessary to be supplemented with the precursor amino acids 508 tyrosine or DOPA to produce HT, a biological system designed to overproduce tyrosine was further 509 engineered by introducing the pS4 and pY3 plasmids to BL21 and HMS174 hosts, for the production 510 of HT directly from glucose (thus named BL21-HT OP or HMS174-HT OP , Table 2). However, 511 preliminary experiments with BL21-HT OP and HMS174-HT OP showed non-consistent tyrosine 512 production, while HT levels remained similar reaching the level of 0.21mM, when both strains 513 evaluated on M9 medium (Fig S5). This was much an unexpected result, since there was a noticeable 514 difference on tyrosine pool, between the two strains. In HMS174-HT OP tyrosine levels reached 523 crispum (Table 1) with respect to the amino acid that accepts as substrate. It is able to convert tyrosine 524 or DOPA to 4-hydroxyphenylacetaldehyde (HPAA, monophenol route) or 3,4-525 dihydroxyphenylacetaldehyde (DHPAA, diphenol route) respectively (Fig 1). The next step should be 526 the reduction of the phenylacetaldehyde to the corresponding alcohol, tyrosol or HT (Table 1, Fig 1).  (Table 1), caused a 62% 534 decrease in the production of HT, it turned to 386% increase when the host was the HMS174 535 (HMS174-HT OP -yahK, Table 1) Table 1), was further analyzed by a LC-MS-MS mass 553 spectrometry. In this highest titer strain the precursor of HT pathway, tyrosine (Fig 1) was internally 554 produced by feeding the strain with glucose supplemented to the growth medium. This strain grown 555 under the protocol Μ9, showed the highest HT production when samples were analyzed from 50ml 556 cultures (Fig 7A) with a CE-DAD system. However, it was important to show whether there was any 557 effect at HT production on large growth medium volume and analyzed by mass spectroscopy. Indeed, 558 the MS analysis showed HT production at much higher levels.
559 After the cleaning and enrichment process of the strain HMS174-HT OP -ALR-K using adsorption 560 resins, the dried weigh of the methanol extract was estimated at 4.2 mg/ml cell culture. In order to 561 determine the HT levels HPLC-ESI(-)-HRMS was employed. 577 coli to produce HT. We chose a rational approach for its production [16] where the primary metabolism 578 was engineered in order to boost tyrosine production that is subsequently utilized for the biosynthesis 579 of HT through the engineered secondary metabolic machinery.
580 The HT molecule bears a benzene (phenolic) ring linked with an alcoholic chain with two carbon atoms 581 ( Fig S4). Since, all phenolic compounds encountered in organisms emanate from the aromatic amino 582 acids via the shikimic acid pathway [48, 49], we assumed that this is also the case for HT biosynthesis.