Biotranformations of natural and inexpensive platform feedstocks to high added value flavour compounds

Lead Research Organisation: Northumbria University
Department Name: Fac of Health and Life Sciences

Abstract

The project focuses on the biocatalysis of relatively inexpensive and abundant natural monoterpenes using previously derived enzyme panels of P450s and KREDS. In particular, alpha pinene (75-90% of turpentine oil) could be transformed to verbenol, verbenone, myrtenol and myrtenal while beta-pinene could be transformed to pinocarveol and pinocarvone. Verbenol and verbenone have been previously produced in microbial systems with black yeast (Hormonema sp) or Aspergilus niger. However, those processes have not been commercialized due to, in the first case interferences with the metabolism of the microorganism and in the latter the commercially non-viable yields. (R)-Limonene is another inexpensive natural monoterpene that has been used as the starting material in the chemical synthesis of carvone, a key volatile compound of caraway seeds, dill seeds and spearmint. Such chemical synthesis could be replaced by biocatalysis using P450s and KRED panels. Other inexpensive naturally derived feedstocks include terpinolene, delta-3-carene, caryophyllene and a-terpineol. Of particular interest is the production of beta-caryophyllene alcohol, a major constituent of all spice, and caryophyllene oxide from beta-caryophyllene as well as carveol from alpha-terpineol. Other approaches will focus on the production of linear terpenals from the corresponding terpenes for example alpha-sinensal from farnesene. The latter is a relatively expensive starting material, however currently there is no other process for the commercial production of sinensal other than distillation from essential oils that contain low amounts of the compound.
Carotenoids have been linked with the formation of a number of volatile compounds in plants (i.e beta ionone, (alpha, beta) damascone, damascenone etc), however, very few studies exist that make use of such feedstocks for the production of flavour molecules. It is known that carotenoids could be degraded by exoenzymes of certain fungi and yeasts leading to the formation of flavour compounds. Trametes versicolor, Marasmius scorodonius and Ischnoderma benzoinum have been shown to result in carotene degradation of 93-98 % while the most abundant flavour volatile identified was beta-ionone (up to 10% conversion). More recently a series of carotenoid cleavage dioxygenases (CCDs) have been isolated from a number of sources, including algae. Central cleavage of beta-carotene catalyzed by beta carotene oxygenase 1 yields two molecules of retinaldehyde, while eccentric cleavage of beta carotene at non-central double bonds is catalyzed by other enzymes and can also occur non-enzymatically (through oxidative cleavage with potassium permanganate). VP14 from maize cleaves 9-(Z) epoxy carotenoids specifically at the 11,12 double bond while other regiospecific carotenoid degrading enzymes have recently been characterised in tomato and petunia flowers. Trichosporon asahii, Paenibacillus amylolyticus, Peurotus eryngii as well as Geotrichum sp were also shown to degrade lutein leading to norisoprenoid flavour compounds with 7, 8-dihydro-beta-ionol and beta ionone as the main products. Versatile peroxidases are a novel class of peroxidases which combine the catalytic properties of lignin peroxidase and manganese peroxidase, typically found in Basidiomycetes. Non volatile products of these reactions, apocarotenals and apocarotenones, could also form platform molecules for the production of a number of sesquiterpenes such as alpha and beta sinensal. Most of the above compounds as well as the resulting end products are hydrophobic molecules and relatively prone to oxidation, however, through previous participation in a feasibility project that aimed to produce aldehydes and alcohols from linolenic acid have developed workflows that are also pertinent to this project while 96 well plate fast screening approaches are also routinely employed.

Technical Summary

The project is divided into the following distinctive work packages and objectives:
a) Cloning & screening: We will perform screening experiments of already characterised P450s and KRED panels to identify the best candidates for the bioconversion of at least 6 platform monoterpenes. Enzymes showing activities for individual substrates will be further explored in small batch bioreactors and analysed via GC/TOF-MS to identify and subsequently quantify the flavour molecules produced. Samples will also be analysed by GC Olfactometry for any unidentified molecules and odour characterisation. Commercially available carotenoids will be explored as potential platform molecules for biotransformations using available CCDs, P450s and KREDS. Lycopene, lutein and astaxanthin will be amongst the tested compounds while identification and characterisation of products will be performed by fractionation using prep HPLC and subsequent LC/MS and NMR. Output of this first work package will be a database of activity vs enzyme identity vs substrate vs products. Any unidentified products that require extra resources will be recorded although as compound identification is a relative laborious process only limited identification exercises will be performed which will be informed by GC-O.
b) Optimise yields using standard enzyme kinetics approaches. The best candidates from the screening sets will be then subjected to enzyme kinetics experiments to optimise yields. This will initially be performed in small batches of circa 1-2 mL and samples analysed by GC MS and GC-O to inform the later stages of the project that relate to the financial viability of the proposed process. Outputs of this work package will be the determination of the reaction conditions that lead to optimum performance for each individual substrate and as a result a database will be created of enzymes vs conditions vs substrate vs products.
c)1 L bench scale fermentations of selected substrate/ enzyme conditions.

Planned Impact

As described in proposal submitted to Innovate UK

Publications

10 25 50
 
Description The research developed novel screening tools for the assessment of biotransformations of relatively inexpensive feedsctocks to high value chemicals. A number of new enzymes were developed and screened against specific substrates pertinent to the flavour industry. Although the novel recombinant enzymes were successfully produced, there was limited activity towards target molecules. Nevertheless, the methodologies and workflows developed in the research are useful for both the University and our industrial partner while the research activities increased the specialist skill ;capability of both our industrial partner and the research base at the University. A bio bank of 50 enzymes pertinent to the project is now available at Northumbria University.
Exploitation Route Through knowledge transfer activities including secondment of staff our industrial partner has gained insight in a research field that is gaining momentum. Scientific community impact will be realised through the publication of improved methodologies and screening tools. The project was a vfeasibility study with limited success on bioconversions that make commercial sense, hence there will be at this stage no commercial impact. Societal impact is realised by increasing the capabilities of the UK bioeconomy workforce leading to socioeconomic growth though the investment of R&D capital increased knowhow.
Sectors Agriculture, Food and Drink,Manufacturing, including Industrial Biotechology,Pharmaceuticals and Medical Biotechnology

 
Title Development of an Improved HPLC methodology for the analysis of carotene and lycopene 
Description HPLC method was performed on an analytical HPLC Ultimate 3000 (Waters, USA). Separation was operated on a Sigma SUPLEX PKB-100 amide column (5┬Ám particle size, 250 mm length x 4.6 mm inner diameter). Isocratic elution was used at a flow rate of 1.5 ml/min. Injection volume was 5 µl. Column temperature was set at 30°C and the autosampler temperature was at 4 °C. The eluting peaks were monitored at 474, 454, 448 and 286 nm using UV-VIS detection. After investigating different parameters, the shortest run time with the optimal resolution was achieved when using 90% mobile phase A (1:1 ACN: MeOH containing 50 ppm BHT in 20 ml 2-propanol, 0.2 ml N-ethyldiisopropylamine, and 50 ppm ammonium acetate dissolved in 25 ml water) and 10% MTBE in the isocratic run of 1.5 ml/min; the injection solvent was MTBE and the injection volume was 5 µl; the column temperature was 30 degrees Celsius. 
Type Of Material Technology assay or reagent 
Year Produced 2017 
Provided To Others? No  
Impact The method has been now part of the methodology portfolio of the group and has been used in other projects including providing services to industrial partners. 
 
Title Fast Screening assays for Determination of enzymatic activity 
Description Fast Screening tests were developed using high throughput LC MS to determine the relationships between substrate and product 
Type Of Material Technology assay or reagent 
Year Produced 2016 
Provided To Others? No  
Impact The methods are now routinely used in our laboratory for the assessment of substrate product relationships in other enzymatic systems 
 
Title Preparation HPLC for the separation and fractionation of lycopene and beta-carotene from raw materials 
Description We have developed and successfully applied a preparative HPLC methodology for the separation and fractionation of lycopene and beta carotene from materials of plant origin. The method uses a preparative HPLC system and a C18 preparative column to separate and subsequently fractionate the carotenoids for downstream testing. The method uses a gradient of methanol and MTBE and separation is achieved within 11 minutes. 
Type Of Material Technology assay or reagent 
Year Produced 2017 
Provided To Others? No  
Impact The method has been since used to fractionate the two compounds present in tomatoes in another project. An SOP was created and we anticipate that the method will be published in the near future. 
 
Title Enzyme database 
Description A database containing 30 recombinant P450s and 30 recombinant CCOs 
Type Of Material Database/Collection of data 
Year Produced 2017 
Provided To Others? No  
Impact The novel recpombinant enzymes coudl be used in other platform molecues to test for activity. Moreover enxyme optimisation coudl be perfomed in silico. 
 
Description Terpene Biotransformations 
Organisation Tree Aid
Country United Kingdom 
Sector Charity/Non Profit 
PI Contribution Provision of knowhow on Flavour Biochemistry, Biotechnology and molecular biology
Collaborator Contribution Provision of objectives and project management. Provision of instrumentation and experimentation
Impact Project to start in April 2016
Start Year 2016