Bio-inspired Chemical Space Exploration of Terpenoids

Many computational methods are used to expand the open-ended border of chemical spaces. Natural products and their derivatives are an important source for drug discovery, and some algorithms are devoted to rapidly generating pseudo-natural products, while their accessibility and chemical interpretation were often ignored or underestimated, thus hampering experimental synthesis in practice. Herein, a bio-inspired strategy (named TeroGen) is proposed, in which the cyclization and decoration stage of terpenoid biosynthesis were mimicked by meta-dynamics simulations and deep learning models respectively, to explore their chemical space. In the protocol of TeroGen, the synthetic accessibility is validated by reaction energetics (reaction barrier and reaction heat) based on the GFN2-xTB methods. Chemical interpretation is an intrinsic feature as the reaction pathway is bioinspired and triggered by the RMSD-PP method in conjunction with an encoder-decoder architecture. This is quite distinct from conventional library/fragment-based or rule-based strategies, by using TeroGen, new reaction routes are feasibly explored to increase the structural diversity. For example, only a rather limited number of sesterterpenoids in our training set is included in this work, but our TeroGen would predict more than 30000 sesterterpenoids and map out the reaction network with super efficiency, ten times as many as the known sesterterpenoids (less than 2500). In sum, TeroGen not only greatly expands the chemical space of terpenoids but also provides various plausible biosynthetic pathways, which are crucial clues for heterologous biosynthesis, bio-mimic and chemical synthesis of complicated terpenoids.

Indirect Pathway Metabolic Engineering Strategies for Enhanced Biosynthesis of Hyaluronic Acid in Engineered Corynebacterium glutamicum

Hyaluronic acid (HA) is composed of alternating d-glucuronic acid and N-acetyl-d-glucosamine, with excellent biocompatibility and water retention capacity. To achieve heterologous biosynthesis of HA, Corynebacterium glutamicum, a safe GRAS (generally recognized as safe) host, was utilized and metabolically engineered previously. In this work, to achieve further enhancement of HA yield, four strategies were proposed and performed separately first, i.e., (1) improvement of glucose uptake via iolR gene knockout, releasing the inhibition of transporter IolT1/IolT2 and glucokinases; (2) intensification of cardiolipin synthesis through overexpression of genes pgsA1/pgsA2/cls involved in cardiolipin synthesis; (3) duly expressed Vitreoscilla hemoglobin in genome, enhancing HA titer coupled with more ATP and improved NAD+/NADH (>7.5) ratio; and (4) identification of the importance of glutamine for HA synthesis through transcriptome analyses and then enhancement of the HA titer via its supplement. After that, we combined different strategies together to further increase the HA titer. As a result, one of the optimal recombinant strains, Cg-dR-CLS, yielded 32 g/L of HA at 60 h in a fed-batch culture, which was increased by 30% compared with that of the starting strain. This high value of HA titer will enable the industrial production of HA via the engineered C. glutamicum.

Functional analysis of Hypoxylon pulicicidum genes required for heterologous biosynthesis of nodulisporic acids

<p>Nature holds some of the greatest secrets in drug design and development and the ability to access these trade secrets has been revolutionised by modern bioengineering technologies. In order to exploit these technologies it is essential to understand what genes are involved in compound production and the enzymatic steps that limit flux to the desired product. This thesis describes the discovery of four secondary-metabolic enzymatic steps involved in the biosynthesis of a group of valuable natural products known as nodulisporic acids. Nodulisporic acids are known for their potent insecticidal activities; however, biosynthesis of these compounds by the natural fungal producer, Hypoxylon pulicicidum (Nodulisporium sp.), is exceptionally difficult and has prevented the commercial development of novel nodulisporic acid-containing veterinary medicines and crop protects. To discover how nodulisporic acids are biosynthesized: 1. the H. pulicicidum genome was sequenced 2. a gene cluster responsible for nodulisporic acid production was predicted 3. genes in the cluster were functionally characterised by pathway reconstitution in a common, fast growing mould, Penicillium paxilli In turn, four genes involved in the biosynthesis of the nodulisporic acid core compound, nodulisporic acid F, have been functionally characterised. The four genes encode a geranylgeranyl transferase (NodC), a flavin adenine dinucleotide-dependent oxygenase (NodM), an indole diterpene cyclase (NodB) and a cytochrome P450 oxygenase (NodW). Two of the gene products (NodM and NodW) catalyse two previously unreported reactions that provide the enzymatic basis of the biosynthetic branch point unique to nodulisporic acid biosynthesis. From here, future efforts will explore how these genes can be engineered to overcome flux bottlenecks and enable production of significantly increased, and even industrially relevant, product titres.</p>

Functional analysis of Hypoxylon pulicicidum genes required for heterologous biosynthesis of nodulisporic acids

<p>Nature holds some of the greatest secrets in drug design and development and the ability to access these trade secrets has been revolutionised by modern bioengineering technologies. In order to exploit these technologies it is essential to understand what genes are involved in compound production and the enzymatic steps that limit flux to the desired product. This thesis describes the discovery of four secondary-metabolic enzymatic steps involved in the biosynthesis of a group of valuable natural products known as nodulisporic acids. Nodulisporic acids are known for their potent insecticidal activities; however, biosynthesis of these compounds by the natural fungal producer, Hypoxylon pulicicidum (Nodulisporium sp.), is exceptionally difficult and has prevented the commercial development of novel nodulisporic acid-containing veterinary medicines and crop protects. To discover how nodulisporic acids are biosynthesized: 1. the H. pulicicidum genome was sequenced 2. a gene cluster responsible for nodulisporic acid production was predicted 3. genes in the cluster were functionally characterised by pathway reconstitution in a common, fast growing mould, Penicillium paxilli In turn, four genes involved in the biosynthesis of the nodulisporic acid core compound, nodulisporic acid F, have been functionally characterised. The four genes encode a geranylgeranyl transferase (NodC), a flavin adenine dinucleotide-dependent oxygenase (NodM), an indole diterpene cyclase (NodB) and a cytochrome P450 oxygenase (NodW). Two of the gene products (NodM and NodW) catalyse two previously unreported reactions that provide the enzymatic basis of the biosynthetic branch point unique to nodulisporic acid biosynthesis. From here, future efforts will explore how these genes can be engineered to overcome flux bottlenecks and enable production of significantly increased, and even industrially relevant, product titres.</p>

Heterologous Biosynthesis and Genomics-Driven Derivatization of Fungal Bioactive Sesterterpenoid Variecolin

The biosynthetic gene cluster of fungal bioactive sesterterpenoids, variecolin (1) and variecolactone (2), was identified in Aspergillus aculeatus ATCC 16872. Heterologous production of 1 and 2 was achieved in Aspergillus oryzae by expressing the sesterterpene synthase VrcA and the cytochrome P450 VrcB. Intriguingly, the replacement of VrcB with homologous P450s from other fungal terpenoid pathways yielded three new variecolin analogues, one of which exhibited potent anticancer activity comparable to that of 1.

Characterisation and heterologous biosynthesis of burnettiene A, a new polyene-decalin polyketide from Aspergillus burnettii

Chemical exploration of the recently described Australian fungus, Aspergillus burnettii, uncovered a new metabolite, burnettiene A. Here, we characterise the structure of burnettiene A as a polyene-decalin polyketide. Bioinformatic analysis of the genome of A. burnettii identified a putative biosynthetic gene cluster for burnettiene A (bue), consisting of eight genes and sharing similarity to the fusarielin gene cluster. Introduction of the reassembled bue gene cluster into Aspergillus nidulans for heterologous expression resulted in the production of burnettiene A under native promoters. Omission of bueE encoding a cytochrome P450 led to the production of preburnettiene A, confirming that BueE is responsible for catalysing the regiospecific multi-oxidation of terminal methyl groups to carboxylic acids. Similarly, bueF was shown to encode an ester-forming methyltransferase, with its omission resulting in the production of the tricarboxylic acid, preburnettiene B. Introduction of an additional copy of the transcription factor bueR under the regulation of the gpdA promoter significantly improved the heterologous production of the burnettienes. Burnettiene A displayed strong in vitro cytotoxicity against mouse myeloma NS-1 cells (MIC 0.8 µg/mL).

Directed yeast genome evolution by controlled introduction of trans-chromosomic structural variations

Background: Naturally occurring structural variations (SVs) are a considerable source of genomic variation and can reshape chromosomes 3D architecture. The synthetic chromosome rearrangement and modification by loxP-mediated evolution (SCRaMbLE) system has been proved to generate random SVs to impact phenotypes and thus constitutes powerful drivers of directed genome evolution. However, controllable methods to introduce complex SVs and revealing the related molecular mechanism has so far remained challenging. Results: We develop a SV-prone yeast strain by using SCRaMbLE with two synthetic chromosomes, synV and synX. An heterologous biosynthesis pathway allowing a high throughput screen for increased yield of astaxanthin is used as readout and a proof of concept for the application of SV in industry. We report here that complex SVs, including a pericentric inversion and a trans-chromosomes translocation between synV and synX, result in two neochromosomes and a 2.7-fold yield of astaxanthin. We mapped genetic targets contributing to higher astaxanthin yield and demonstrated the SVs can led to large reorganization of the genetic information along the chromosomes. We also used the model learned from the aforementioned random screen and successfully harnessed the precise introduction of trans-chromosomes translocation and pericentric inversions by rational design. Conclusions: Our work provides an effective tool to not only accelerate the directed genome evolution but also reveal mechanistic insight of complex SVs for altering phenotypes.

Heterologous Biosynthesis of Artemisinin in Chrysanthemum morifolium Ramat

Artemisinin-based drugs are the most effective medicine against multidrug-resistant Plasmodium spp., the parasite that causes malaria. To this day, wormwood A. annua L. is the sole commercial source of artemisinin, where it is produced in minor amounts. The artemisinin yield depends on numerous poorly regulated agricultural factors and the genetic variability of this non-domesticated plant. This has aroused significant interest in the development of heterologous expression platforms for artemisinin production. Previously, we obtained lines of Chrysanthemum morifolium Ramat. (C. morifolium Ramat.), cvs. White Snowdon and Egyptianka, transformed with artemisinin biosynthesis genes. Here, we report the results of an analysis of artemisinin production in transgenic chrysanthemums. Transcription of heterologous amorpha-4,11-diene monooxygenase and cytochrome P450 reductase genes in transgenic lines was confirmed using high-resolution melting analysis. Artemisinin accumulation was detected using GC-MS in White Snowdon plants, but not in Egyptianka ones, thereby demonstrating the possibility of transplanting active artemisinin biosynthetic pathway into chrysanthemum. Ways of increasing its content in producer plants are discussed.