Biosynthetic Engineering Research Articles

Site-Directed Mutagenesis of Modular Polyketide Synthase Ketoreductase Domains for Altered Stereochemical Control.

Bacterial modular type I polyketide synthases (PKSs) are complex multidomain assembly line proteins that produce a range of pharmaceutically relevant molecules with a high degree of stereochemical control. Due to their colinear properties, they have been considerable targets for rational biosynthetic pathway engineering. Among the domains harbored within these complex assembly lines, ketoreductase (KR) domains have been extensively studied with the goal of altering their stereoselectivity by site-directed mutagenesis, as they confer much of the stereochemical complexity present in pharmaceutically active reduced polyketide scaffolds. Here we review all efforts to date to perform site-directed mutagenesis on PKS KRs, most of which have been done in the context of excised KR domains on model diffusible substrates such as β-keto N-acetyl cysteamine thioesters. We also discuss the challenges around translating the findings of these studies to alter stereocontrol in the context of a complex multidomain enzymatic assembly line.

Functional expression of eukaryotic cytochrome P450s in yeast.

Cytochrome P450 enzymes (P450s) are a superfamily of heme-thiolate proteins widely existing in various organisms. Due to their key roles in secondary metabolism, degradation of xenobiotics, and carcinogenesis, there is a great demand to heterologously express and obtain a sufficient amount of active eukaryotic P450s. However, most eukaryotic P450s are endoplasmic reticulum-localized membrane proteins, which is the biggest challenge for functional expression to high levels. Furthermore, the functions of P450s require the cooperation of cytochrome P450 reductasesfor electron transfer. Great efforts have been devoted to the heterologous expression of eukaryotic P450s, and yeasts, particularly Saccharomyces cerevisiae are frequently considered as the first expression systems to be tested for this challenging purpose. This review discusses the strategies for improving the expression and activity of eukaryotic P450s in yeasts, followed by examples of P450s involved in biosynthetic pathway engineering.

Formation and characteristics of soot from pyrolysis of ethylene blended with furan fuels

As two renewable oxygenated biofuels, 2,5-dimethylfuran and 2-methylfuran (DMF and MF) have been considered to be two of the most potential fuels in the future due to the development of the second-generation biosynthetic technologies. The atmosphere pyrolysis experiments with 0%, 25%, 50%, 75% and 100% replacement of ethylene by DMF/MF at 1173 and 1273 K were conducted. Collected soot samples were characterized by high resolution transmission electron microscopy (HRTEM) and thermogravimetric analysis (TGA) to acquire their internal structure and oxidation reactivity. Results showed that soot mass was positively related with DMF addition ratios and the reaction temperature. Soot production was also enhanced when the MF addition ratio gradually increased from 0% to 75%. The influences of DMF addition can promote soot formation more obviously than MF. Temperatures showed a more significant influence on soot morphology than fuel types and DMF/MF addition ratios. For a fixed addition ratio of DMF/MF, soot showed liquid-like substances at 1173 K. At 1273 K, approximately round particles formed and linked together in chains. Moreover, at 1273 K, the aggregates obtained by adding MF contained more single particles, longer carbon chains, and larger projection area compared with the aggregates by adding same MF. For nanostructures, as the addition ratios of DMF/MF increased, as well as the reaction temperature improved, the fringe length of the carbon layer increased, the average fringe tortuosity decreased, and the stacking arrangement of soot became more orderly, the soot oxidation reactivity was lower. Under a same addition ratio and reaction temperature, soot obtained by adding DMF possessed slightly longer fringe length, smaller fringe tortuosity and lower oxidation reactivity than those of the soot obtained by adding MF. High correlation between fringe parameters and soot oxidation reactivity was discovered. The more ordered soot nanostructure, the longer fringe length, and the smaller fringe tortuosity could make the oxidation reactivity of soot get lower.

Biotechnological production of lycopene by microorganisms.

Lycopene is a dark red carotenoid belonging to C40 terpenoids and is widely found in a variety of plants, especially ripe red fruits and vegetables. Lycopene has been shown to reduce the risk of prostate cancer, other cancers, and cardiovascular disease. It is one of the most widely used carotenoids in the healthcare product market. Currently, commercially available lycopene is mainly extracted from tomatoes. However, production of lycopene from plants is costly and environmentally unfriendly. To date, there have been many reports on the biosynthesis of lycopene by microorganisms, providing another route for lycopene production. This review discusses the lycopene biosynthetic pathway and natural and engineered lycopene-accumulating microorganisms, as well as their production of lycopene. The effects of different metabolic engineering strategies on lycopene accumulation are also considered. Furthermore, this work presents perspectives concerning the microbial production of lycopene, especially trends to construct microbial cell factories for lycopene production. KEY POINTS: • Recent achievements in the lycopene biosynthesis in microorganisms. • Review of lycopene biosynthetic metabolism engineering strategy. • Discuss the current challenges and prospects of using microorganisms to produce lycopene.

Halogenases for biosynthetic pathway engineering: Toward new routes to naturals and non-naturals

ABSTRACT Nature’s repertoire of bio-halogenase enzymes is intriguing with halogenases from various natural product biosynthetic clusters that carry out site and region-specific halogenation of diverse bioactive precursors and molecules. Currently, we have a comprehensive catalogue of cryptic and non-cryptic halogenases that act on simple to complex aliphatic and aromatic molecular scaffolds. This will open up further synthetic and biosynthetic opportunities for C-H activation, ring formation and functionalization of different molecular structures. In fact, halogenases were exploited over the years for these potential applications, to replace traditional chemical halogenation chemistries toward creating economical and environmentally benign methodologies and also for biosynthetic pathways. This review will discuss our advances in utilizing bio-halogenases to generate both in vivo and in vitro biosynthetic pathways; summarizing all naturals and non-naturals that are synthesized with a direct bio-halogenase incorporation.

Synthetic biology, combinatorial biosynthesis, and chemo‑enzymatic synthesis of isoprenoids.

Isoprenoids are a large class of natural products with myriad applications as bioactive and commercial compounds. Their diverse structures are derived from the biosynthetic assembly and tailoring of their scaffolds, ultimately constructed from two C5 hemiterpene building blocks. The modular logic of these platforms can be harnessed to improve titers of valuable isoprenoids in diverse hosts and to produce new-to-nature compounds. Often, this process is facilitated by the substrate or product promiscuity of the component enzymes, which can be leveraged to produce novel isoprenoids. To complement rational enhancements and even re-programming of isoprenoid biosynthesis, high-throughput approaches that rely on searching through large enzymatic libraries are being developed. This review summarizes recent advances and strategies related to isoprenoid synthetic biology, combinatorial biosynthesis, and chemo-enzymatic synthesis, focusing on the past 5 years. Emerging applications of cell-free biosynthesis and high-throughput tools are included that culminate in a discussion of the future outlook and perspective of isoprenoid biosynthetic engineering.

Mining Symbionts of a Spider-Transmitted Fungus Illuminates Uncharted Biosynthetic Pathways to Cytotoxic Benzolactones.

A spider‐transmitted fungus (Rhizopus microsporus) that was isolated from necrotic human tissue was found to harbor endofungal bacteria (Burkholderia sp.). Metabolic profiling of the symbionts revealed a complex of cytotoxic agents (necroximes). Their structures were characterized as oxime‐substituted benzolactone enamides with a peptidic side chain. The potently cytotoxic necroximes are also formed in symbiosis with the fungal host and could have contributed to the necrosis. Genome sequencing and computational analyses revealed a novel modular PKS/NRPS assembly line equipped with several non‐canonical domains. Based on gene‐deletion mutants, we propose a biosynthetic model for bacterial benzolactones. We identified specific traits that serve as genetic handles to find related salicylate macrolide pathways (lobatamide, oximidine, apicularen) in various other bacterial genera. Knowledge of the biosynthetic pathway enables biosynthetic engineering and genome‐mining approaches.

Mining Symbionts of a Spider‐Transmitted Fungus Illuminates Uncharted Biosynthetic Pathways to Cytotoxic Benzolactones

AbstractA spider‐transmitted fungus (Rhizopus microsporus) that was isolated from necrotic human tissue was found to harbor endofungal bacteria (Burkholderia sp.). Metabolic profiling of the symbionts revealed a complex of cytotoxic agents (necroximes). Their structures were characterized as oxime‐substituted benzolactone enamides with a peptidic side chain. The potently cytotoxic necroximes are also formed in symbiosis with the fungal host and could have contributed to the necrosis. Genome sequencing and computational analyses revealed a novel modular PKS/NRPS assembly line equipped with several non‐canonical domains. Based on gene‐deletion mutants, we propose a biosynthetic model for bacterial benzolactones. We identified specific traits that serve as genetic handles to find related salicylate macrolide pathways (lobatamide, oximidine, apicularen) in various other bacterial genera. Knowledge of the biosynthetic pathway enables biosynthetic engineering and genome‐mining approaches.

Generation of tetramycin B derivative with improved pharmacological property based on pathway engineering.

Polyene antibiotics, including amphotericin, nystatin, pimaricin, and tetramycin, are important antifungal agents. Increasing the production of polyenes and generation of their improved analogues based on the biosynthetic pathway engineering has aroused wide concern in application researches. Herein, tetramycin and nystatin, both of which share most of acyl-CoA precursors, are produced by Streptomyces hygrospinosus var. beijingensis CGMCC 4.1123. Thus, the intracellular malonyl-CoA is found to be insufficient for PKSs (polyketide synthases) extension of tetramycin by quantitative analysis in this wild-type strain. To circumvent this problem and increase tetramycin titer, the acyl-CoA competing biosynthetic gene cluster (BGC) of nystatin was disrupted, and the biosynthetic genes of malonyl-CoA from S. coelicolor M145 were integrated and overexpressed in nys-disruption mutant strain (SY02). Moreover, in order to specifically accumulate tetramycin B from A, two copies of tetrK and a copy of tetrF were introduced, resulting in elevating tetramycin B fermentrationtiter by 122% to 865 ± 8mg/L than the wild type. In this optimized strain, a new tetramycin derivative, 12-decarboxy-12-methyl tetramycin B, was generated with a titer of 371 ± 26mg/L through inactivation of a P450 monooxygenase gene tetrG. Compared with tetramycin B, the new compound exhibited higher antifungal activity against Saccharomyces cerevisiae and Rhodotorula glutinis, but lower hemolytic toxicity to erythrocyte. This research provided a good example of employing biosynthetic engineering strategies for fermentation titer improvement of polyene and development of the derivatives for medicinal applications.

Biosynthetic engineering of the antifungal, anti-MRSA auroramycin

Using an established CRISPR-Cas mediated genome editing technique for streptomycetes, we explored the combinatorial biosynthesis potential of the auroramycin biosynthetic gene cluster in Streptomyces roseosporous. Auroramycin is a potent anti-MRSA polyene macrolactam. In addition, auroramycin has antifungal activities, which is unique among structurally similar polyene macrolactams, such as incednine and silvalactam. In this work, we employed different engineering strategies to target glycosylation and acylation biosynthetic machineries within its recently elucidated biosynthetic pathway. Auroramycin analogs with variations in C-, N- methylation, hydroxylation and extender units incorporation were produced and characterized. By comparing the bioactivity profiles of five of these analogs, we determined that unique disaccharide motif of auroramycin is essential for its antimicrobial bioactivity. We further demonstrated that C-methylation of the 3, 5-epi-lemonose unit, which is unique among structurally similar polyene macrolactams, is key to its antifungal activity.

Heterologous Biosynthesis of Type II Polyketide Products Using E. coli

The heterologous biosynthesis of complex natural products has enabled access to polyketide, nonribosomal peptide, isoprenoid, and other compounds with wide-spanning societal value. Though several surrogate host systems exist, Escherichia coli is often a preferred choice due to its rapid growth kinetics and extensive molecular biology protocols. However, a persistent challenge to the utilization of E. coli has been the successful in vivo reconstitution of type II polyketide synthase (PKS) systems. In particular, gene expression of the ketosynthase (KS) components of the minimal PKS has consistently yielded insoluble protein products. In the following report, two type II PKS systems were functionally reconstituted in E. coli. The approach to do so relied upon the utilization of the native transcriptional coupling between the dimeric KS subunits, leading to soluble recombinant protein products and successful polyketide biosynthesis. Resulting strains produced 10 mg/L TW95c and 25 mg/L dehydrorabelomycin. Hence, the strategy offers a new option in the biosynthetic engineering efforts for the heterologous production of type II polyketide products using E. coli.

Development of Non-Immunosuppressive FK506 Derivatives as Antifungal and Neurotrophic Agents.

FK506, also known as tacrolimus, is a clinically important immunosuppressant drug and has promising therapeutic potentials owing to its antifungal, neuroprotective, and neuroregenerative activities. To generate various FK506 derivatives, the structure of FK506 has been modified by chemical methods or biosynthetic pathway engineering. Herein, we describe the mode of the antifungal action of FK506 and the structure-activity relationship of FK506 derivatives in the context of immunosuppressive and antifungal activities. In addition, we discuss the neurotrophic mechanism of FK506 known to date, along with the neurotrophic FK506 derivatives with significantly reduced immunosuppressive activity. This review suggests the possibility to generate novel FK506 derivatives as antifungal as well as neuroregenerative/neuroprotective agents.

Off-Colony Screening of Biosynthetic Libraries by Rapid Laser-Enabled Mass Spectrometry.

By leveraging advances in DNA synthesis and molecular cloning techniques, synthetic biology increasingly makes use of large construct libraries to explore large design spaces. For biosynthetic pathway engineering, the ability to screen these libraries for a variety of metabolites of interest is essential. If the metabolite of interest or the metabolic phenotype is not easily measurable, screening soon becomes a major bottleneck involving time-consuming culturing, sample preparation, and extraction. To address this, we demonstrate the use of automated laser-assisted rapid evaporative ionization mass spectrometry (LA-REIMS)-a form of ambient laser desorption ionization mass spectrometry-to perform rapid mass spectrometry analysis direct from agar plate yeast colonies without sample preparation or extraction. We use LA-REIMS to assess production levels of violacein and betulinic acid directly from yeast colonies at a rate of 6 colonies per minute. We then demonstrate the throughput enabled by LA-REIMS by screening over 450 yeast colonies within <4 h, while simultaneously generating recoverable glycerol stocks of each colony in real time. This showcases LA-REIMS as a prescreening tool to complement downstream quantification methods such as liquid chromatography-mass spectroscopy (LCMS). By prescreening several hundred colonies with LA-REIMS, we successfully isolate and verify a strain with a 2.5-fold improvement in betulinic acid production. Finally, we show that LA-REIMS can detect 20 out of a panel of 27 diverse biological molecules, demonstrating the broad applicability of LA-REIMS to metabolite detection. The rapid and automated nature of LA-REIMS makes this a valuable new technology to complement existing screening technologies currently employed in academic and industrial workflows.

Structural basis for chain release from the enacyloxin polyketide synthase.

SummaryModular polyketide synthases and nonribosomal peptide synthetases are molecular assembly lines consisting of several multienzyme subunits that undergo dynamic self-assembly to form a functional mega-complex. N- and C-terminal docking domains are usually responsible for mediating interactions between subunits. Here we show that communication between two nonribosomal peptide synthetase subunits responsible for chain release from the enacyloxin polyketide synthase, which assembles an antibiotic with promising activity against Acinetobacter baumannii, is mediated by an intrinsically disordered short linear motif and a β-hairpin docking domain. The structures, interactions and dynamics of these subunits are characterised using several complementary biophysical techniques, providing extensive insights into binding and catalysis. Bioinformatics analyses reveal that short linear motif/β-hairpin docking domain pairs mediate subunit interactions in numerous nonribosomal peptide and hybrid polyketide-nonribosomal peptide synthetases, including those responsible for assembling several important drugs. Short linear motifs and β-hairpin docking domains from heterologous systems are shown to interact productively, highlighting the potential of such interfaces as tools for biosynthetic engineering.

Biobased lubricant firm raises funds

Biosynthetic Technologies has raised $9.5 million in a first round of funding to commercialize its Estolides line of biobased synthetic oils for use in lubricants, chemicals, and cosmetics. Early-s...

Pentamycin Biosynthesis in Philippine Streptomyces sp. S816: Cytochrome P450-Catalyzed Installation of the C-14 Hydroxyl Group.

Pentamycin is a polyene antibiotic, registered in Switzerland for the treatment of vaginal candidiasis, trichomoniasis, and mixed infections. Chemical instability has hindered its widespread application and development as a drug. Here, we report the identification of Streptomyces sp. S816, isolated from Philippine mangrove soil, as a pentamycin producer. Genome sequence analysis identified the putative pentamycin biosynthetic gene cluster, which shows a high degree of similarity to the gene cluster responsible for filipin III biosynthesis. The ptnJ gene, which is absent from the filipin III biosynthetic gene cluster, was shown to encode a cytochrome P450 capable of converting filipin III to pentamycin. This confirms that the cluster directs pentamycin biosynthesis, paving the way for biosynthetic engineering approaches to the production of pentamycin analogues. Several other Streptomyces genomes were found to contain ptnJ orthologues clustered with genes encoding polyketide synthases that appear to have similar architectures to those responsible for the assembly of filipin III and pentamycin, suggesting pentamycin production may be common in Streptomyces species.

Recent advances in natural products exploitation in Streptomyces via synthetic biology.

Natural products of microbial origin have proven to be the wellspring of clinically useful compounds for human therapeutics. Streptomyces species are predominant sources of bioactive compounds, most of which serve as potential drug candidates. While the exploitation of natural products has been severely reduced over the past two decades, the growing crisis of evolution and dissemination of drug resistant pathogens have again attracted great interest in this field. The emerging synthetic biology has been heralded as a new bioengineering platform to discover novel bioactive compounds and expand bioactive natural products diversity and production. Herein, we review recent advances in the natural products exploitation of Streptomyces with the applications of synthetic biology from three major aspects, including recently developed synthetic biology tools, natural products biosynthetic pathway engineering strategies as well as chassis host modifications.

Computational identification of co-evolving multi-gene modules in microbial biosynthetic gene clusters

The biosynthetic machinery responsible for the production of bacterial specialised metabolites is encoded by physically clustered group of genes called biosynthetic gene clusters (BGCs). The experimental characterisation of numerous BGCs has led to the elucidation of subclusters of genes within BGCs, jointly responsible for the same biosynthetic function in different genetic contexts. We developed an unsupervised statistical method able to successfully detect a large number of modules (putative functional subclusters) within an extensive set of predicted BGCs in a systematic and automated manner. Multiple already known subclusters were confirmed by our method, proving its efficiency and sensitivity. In addition, the resulting large collection of newly defined modules provides new insights into the prevalence and putative biosynthetic role of these modular genetic entities. The automated and unbiased identification of hundreds of co-evolving group of genes is an essential breakthrough for the discovery and biosynthetic engineering of high-value compounds.

Engineering Atypical Tetracycline Formation in Amycolatopsis sulphurea for the Production of Modified Chelocardin Antibiotics.

To combat the increasing spread of antimicrobial resistance and the shortage of novel anti-infectives, one strategy for the development of new antibiotics is to optimize known chemical scaffolds. Here, we focus on the biosynthetic engineering of Amycolatopsis sulphurea for derivatization of the atypical tetracycline chelocardin and its potent broad-spectrum derivative 2-carboxamido-2-deacetyl-chelocardin. Heterologous biosynthetic genes were introduced into this chelocardin producer to modify functional groups and generate new derivatives. We demonstrate cooperation of chelocardin polyketide synthase with tailoring enzymes involved in biosynthesis of oxytetracycline from Streptomyces rimosus. An interesting feature of chelocardin, compared with oxytetracycline, is the opposite stereochemistry of the C4 amino group. Genes involved in C4 transamination and N,N-dimethylation of oxytetracycline were heterologously expressed in an A. sulphurea mutant lacking C4-aminotransferase. Chelocardin derivatives with opposite stereochemistry of the C4 amino group, as N,N-dimethyl- epi-chelocardin and N,N-dimethyl-2-carboxamido-2-deacetyl- epi-chelocardin, were produced only when the aminotransferase from oxytetracycline was coexpressed with the N-methyltransferase OxyT. Surprisingly, OxyT exclusively accepted intermediates carrying an S-configured amino group at C4 in chelocardin. Applying medicinal chemistry approaches, several 2-carboxamido-2-deacetyl- epi-chelocardin derivatives modified at C4 were produced. Analysis of the antimicrobial activities of the modified compounds demonstrated that the primary amine in the R configuration is a crucial structural feature for activity of chelocardin. Unexpectedly, C10 glycosylated chelocardin analogues were identified, thus revealing the glycosylation potential of A. sulphurea. However, efficient glycosylation of the chelocardin backbone occurred only after engineering of a dimethylated amino group at the C4 position in the opposite S configuration, which suggests some evolutionary remains of chelocardin glycosylation.

Unintentional Genomic Changes Endow Cupriavidus metallidurans with an Augmented Heavy-Metal Resistance.

For the past three decades, Cupriavidus metallidurans has been one of the major model organisms for bacterial tolerance to heavy metals. Its type strain CH34 contains at least 24 gene clusters distributed over four replicons, allowing for intricate and multilayered metal responses. To gain organic mercury resistance in CH34, broad-spectrum mer genes were introduced in a previous work via conjugation of the IncP-1β plasmid pTP6. However, we recently noted that this CH34-derived strain, MSR33, unexpectedly showed an increased resistance to other metals (i.e., Co2+, Ni2+, and Cd2+). To thoroughly investigate this phenomenon, we resequenced the entire genome of MSR33 and compared its DNA sequence and basal gene expression profile to those of its parental strain CH34. Genome comparison identified 11 insertions or deletions (INDELs) and nine single nucleotide polymorphisms (SNPs), whereas transcriptomic analysis displayed 107 differentially expressed genes. Sequence data implicated the transposition of IS1088 in higher Co2+ and Ni2+ resistances and altered gene expression, although the precise mechanisms of the augmented Cd2+ resistance in MSR33 remains elusive. Our work indicates that conjugation procedures involving large complex genomes and extensive mobilomes may pose a considerable risk toward the introduction of unwanted, undocumented genetic changes. Special efforts are needed for the applied use and further development of small nonconjugative broad-host plasmid vectors, ideally involving CRISPR-related and advanced biosynthetic technologies.