Understanding carbazole biosynthetic enzymes: potential for a versatile assay of acyl CoAs

Fatty acid coenzyme As (acyl-CoAs) ubiquitously exist in living organisms and play central roles in many biochemical reactions. Abnormal accumulation of acyl-CoAs in cells are often the diagnostic symptoms for many diseases (i.e. human metabolic diseases including diabetes, fatty acid oxidation deficiency (FAOD), and cancers). Considerable efforts have gone into developing potential diagnostic tools to measure acyl CoAs so far and yet the outcomes are far from ideal.
Acyl CoAs are also the key precursors for microbial fermentation of fatty acid-derived chemicals. Currently many of these chemicals are prepared from fossil-derived feedstocks which require energy intensive processing, and are considered by many as non-sustainable for the future. In this respect, microbial fermentation is an attractive alternative to produce these commodity chemicals and enhanced acyl CoA production is associated with increased production of fatty acid derived chemicals in engineered microbes. In making acyl CoA enhanced, a key challenge is often to quantify the levels of acyl CoA among engineered microbes but researchers have yet to find a reliable way of doing this.
In this study, we are going to investigate an enzyme-based system that has the ability to convert acyl-CoAs into coloured aromatic compounds under mild conditions. These aromatic compounds are common motifs in dye materials. Consequently, the incorporation of our enzymatic system into biological samples will offer a versatile and convenient assay to measure acyl CoA concentration within the given samples. The use of bacterial enzymes to accomplish chemical tasks is well established in the food industry. The benefits in terms of sustainable manufacturing are well documented. However, the application of enzymatic processes/industrial biotechnology processes are still underdeveloped in the fine chemical, pharmaceutical, agricultural and bioscience industries. By working out the catalytic capabilities of the two novel enzymes involved in transforming acyl CoAs into coloured aromatic compounds, we will gain the ability to develop their biotechnological potential to be used as a new sensitive assay that will enable us to directly measure acyl CoAs in various biological samples.

We propose to investigate the capabilities of two novel enzymes, a new III ketosynthase (KSIII) (NzsJ) and a new aromatase (NzsI), from Streptomyces sp. MA37, involved in the formation of a carbazole alkaloid (CA). These heterotricyclic molecules are critical components of many biologically important and industrially relevant molecules (i.e. dye materials). We have made significant progress in understanding the chemical mechanisms of two key enzymes (NzsE&H) involved in the biosynthesis of this CA, the first step towards harnessing the biotransformation potential of this group of molecules. From our preliminary results, we have begun to understand the mechanisms of the new KSIII NzsJ and our data indicated the formation of an indole-fused hexanone intermediate. We intend to continue this work and elucidate the mechanism of this enzyme in detail through site-directed mutagenesis studies to locate its active site. We will also probe the capacity of this enzyme using modified substrates with structural diversity. We have also started to understand the mechanism of the novel aromatase NzsI using stable isotopic labelling studies. Our data indicates the formation of the aromatic heterotricyclic ring via a one-pot biotransformation. We will advance our initial mechanistic work by using substrates substituted with various functional groups. Substrate tolerance is a key feature for a successful industrial biocatalyst. We will synthesize acyl thioesters equipped with different chain length and functionalities (i.e. saturated and unsaturated ring systems) and probe the substrate flexibility of NzsJ and NzsI. Furthermore, we will identify new homologues of NzsJ and NzsI that possess enhanced kinetic profiles and broader substrate tolerance through genome mining and synthetic gene expression.
This will not only give us control on the biochemical transformations but also allow us to develop a versatile and efficient colorimetric assay for acylCoA in various biological samples.

Impact will be delivered by fulfilling the research programme's key objective which are to provide stakeholders in various sectors with our new technology.

The project will deliver impact across four main areas.
People: The program will deliver multidisciplinary training in the chemical and biological sciences to one PDRA. The UK has identified synthetic biology, industrial biotechnology and new technology to biosciences as key deficits in scientists' training for the future workforce and this program will address those needs. The project is at the cutting edge of industrial biotechnology and will provide excellent training for the PDRA to ensure the PDRA can develop a career in industry or academia. The PDRA will also have access to a variety of staff training courses run by the University of Aberdeen, which are designed to enhance a wide variety of transferable and career based skills. All of the aforementioned points will ensure that the recruited PDRA has enhanced job prospects upon completion of the program and this will help to enhance the UK's scientific skill base. The PI's (Deng) lab regularly hosts undergraduates from the UK or Europe as summer placement students. Both Deng and Trembleau (departmental recruitment officer) are very active in outreach programmes that engage with secondary school pupils. If funded we will offer summer placements (1-2 per year) to local pupils to experience research at the chemical biology interface. We also intend to create a range of practical biochemical experiments suitable for University undergraduates in the first instance at Aberdeen but that will be made open-access via the PI's webpage.
Education: Interaction with local secondary schools will also help to enthuse the next generation of scientists. The Co-I (Trembleau) has prior experience, in this area, having led various projects as part of recruitment programmes held in the Chemistry Department (i.e. spectroscopy in suitcase, School visits, British Science Week). Students from secondary schools in the North-East of Scotland, accompanied by their teachers, spend a full week undertaking research projects.
Society: The work will offer new technologies to the biosciences using sustainable manufacturing methods (i.e. biocatalysts). The success of the programme will offer diagnostic tools that can be used in screening campaigns for diseases. The early detection of these diseases has the potential to provide a proper evaluation and management in a timely fashion both in the National Health Service (NHS), and, more directly in everyday life. The tools developed in this programme can be also used as an indicator to improve microbial fermentation of commodity chemicals in the fine chemicals. Delivering in these areas will in turn have a direct and potentially considerable impact on the health of the UK population and sustainable future.
Economic: The generation of novel IP will help to develop new markets for commercial exploitation which will help the UK PLC to remain internationally competitive in the areas of chemical-biology, biosciences and industrial biotechnology. As part of the work program we will utilise existing links and aim to establish new collaborations with industry to maximise the commercial exploitation of the research. We would seek to transfer technology through service agreements or by technology licensing. We will consider founding our own spin out company as the project develops.
Knowledge: At a fundamental level the programme will deliver exciting new insights into new enzymes (biocatalysts), NzsJ and NzsI, involved in the biosynthesis of carbazole alkaloids and specifically their mode of action. This new knowledge will be of benefit to researchers both in academia and in industries who are engaged in developing new biotransformation technology to biosciences.