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The tetrahydroisoquinoline (THIQ) moiety is a privileged substructure of many bioactive natural products and semi-synthetic analogs. Plants manufacture more than 3,000 THIQ alkaloids, including the opioids morphine and codeine. While microbial species have been engineered to synthesize a few compounds from the benzylisoquinoline alkaloid (BIA) family of THIQs, low product titers impede industrial viability and limit access to the full chemical space. Here we report a yeast THIQ platform by increasing production of the central BIA intermediate ( S )-reticuline to 4.6 g L −1 , a 57,000-fold improvement over our first-generation strain. We show that gains in BIA output coincide with the formation of several substituted THIQs derived from amino acid catabolism. We use these insights to repurpose the Ehrlich pathway and synthesize an array of THIQ structures. This work provides a blueprint for building diverse alkaloid scaffolds and enables the targeted overproduction of thousands of THIQ products, including natural and semi-synthetic opioids. Plants synthesize more than 3000 tetrahydroisoquinoline (THIQ) alkaloids, but only a few of them have been produced by engineered microbes and titers are very low. Here, the authors increase ( S )-reticuline titer to 4.6 g/L and repurpose the yeast Ehrlich pathway to synthesize a diverse array of THIQ scaffolds.

Saccharomyces cerevisiae is a workhorse of industrial biotechnology owing to the organism’s prominence in alcohol fermentation and the suite of sophisticated genetic tools available to manipulate its metabolism. However, S. cerevisiae is not suited to overproduce many bulk bioproducts, as toxicity constrains production at high titers. Here, we employ a high-throughput assay to screen 108 publicly accessible yeast strains for tolerance to 20 g L −1 adipic acid (AA), a nylon precursor. We identify 15 tolerant yeasts and select Pichia occidentalis for production of cis , cis -muconic acid (CCM), the precursor to AA. By developing a genome editing toolkit for P. occidentalis , we demonstrate fed-batch production of CCM with a maximum titer (38.8 g L −1 ), yield (0.134 g g −1 glucose) and productivity (0.511 g L −1  h −1 ) that surpasses all metrics achieved using S. cerevisiae . This work brings us closer to the industrial bioproduction of AA and underscores the importance of host selection in bioprocessing. Baker’s yeast is a workhorse of industrial biotechnology, but it is not suited to overproduce many bulk bioproducts, especially organic acids. Here, the authors identify Pichia occidentalis as an acid tolerant yeast and engineer it for the production of muconic acid using a newly developed genome editing toolkit.

Application of CRISPR-Cas9 systems has revolutionized genome editing across all domains of life. Here we report implementation of the heterologous Type II CRISPR-Cas9 system in Clostridium pasteurianum for markerless genome editing. Since 74% of species harbor CRISPR-Cas loci in Clostridium , we also explored the prospect of co-opting host-encoded CRISPR-Cas machinery for genome editing. Motivation for this work was bolstered from the observation that plasmids expressing heterologous cas9 result in poor transformation of Clostridium . To address this barrier and establish proof-of-concept, we focus on characterization and exploitation of the C. pasteurianum Type I-B CRISPR-Cas system. In silico spacer analysis and in vivo interference assays revealed three protospacer adjacent motif (PAM) sequences required for site-specific nucleolytic attack. Introduction of a synthetic CRISPR array and cpaAIR gene deletion template yielded an editing efficiency of 100%. In contrast, the heterologous Type II CRISPR-Cas9 system generated only 25% of the total yield of edited cells, suggesting that native machinery provides a superior foundation for genome editing by precluding expression of cas9 in trans . To broaden our approach, we also identified putative PAM sequences in three key species of Clostridium . This is the first report of genome editing through harnessing native CRISPR-Cas machinery in Clostridium .

An overview of common challenges and strategies underlying efforts to reconstruct plant isoprenoid, alkaloid, phenylpropanoid, and polyketide biosynthetic pathways in microbial systems.

Doc number: 50 Abstract Background: Reducing the production cost of, and increasing revenues from, industrial biofuels will greatly facilitate their proliferation and co-integration with fossil fuels. The cost of feedstock is the largest cost in most fermentation bioprocesses and therefore represents an important target for cost reduction. Meanwhile, the biorefinery concept advocates revenue growth through complete utilization of by-products generated during biofuel production. Taken together, the production of biofuels from low-cost crude glycerol, available in oversupply as a by-product of bioethanol production, in the form of thin stillage, and biodiesel production, embodies a remarkable opportunity to advance affordable biofuel development. However, few bacterial species possess the natural capacity to convert glycerol as a sole source of carbon and energy into value-added bioproducts. Of particular interest is the anaerobe Clostridium pasteurianum , the only microorganism known to convert glycerol alone directly into butanol, which currently holds immense promise as a high-energy biofuel and bulk chemical. Unfortunately, genetic and metabolic engineering of C. pasteurianum has been fundamentally impeded due to lack of an efficient method for deoxyribonucleic acid (DNA) transfer. Results: This work reports the development of an electrotransformation protocol permitting high-level DNA transfer to C. pasteurianum ATCC 6013 together with accompanying selection markers and vector components. The CpaAI restriction-modification system was found to be a major barrier to DNA delivery into C. pasteurianum which we overcame by in vivo methylation of the recognition site (5'-CGCG-3') using the M.FnuDII methyltransferase. With proper selection of the replication origin and antibiotic-resistance marker, we initially electroporated methylated DNA into C. pasteurianum at a low efficiency of 2.4 × 101 transformants μg-1 DNA by utilizing conditions common to other clostridial electroporations. Systematic investigation of various parameters involved in the cell growth, washing and pulse delivery, and outgrowth phases of the electrotransformation procedure significantly elevated the electrotransformation efficiency, up to 7.5 × 104 transformants μg-1 DNA, an increase of approximately three order of magnitude. Key factors affecting the electrotransformation efficiency include cell-wall-weakening using glycine, ethanol-mediated membrane solubilization, field strength of the electric pulse, and sucrose osmoprotection. Conclusions: C. pasteurianum ATCC 6013 can be electrotransformed at a high efficiency using appropriately methylated plasmid DNA. The electrotransformation method and tools reported here should promote extensive genetic manipulation and metabolic engineering of this biotechnologically important bacterium.

We recently reported the heterologous production of 1-propanol in Escherichia coli via extended dissimilation of succinate under anaerobic conditions through expression of the endogenous sleeping beauty mutase (Sbm) operon. In the present work, we demonstrate high-level coproduction of 1-propanol and ethanol by developing novel engineered E. coli strains with effective cultivation strategies. Various biochemical, genetic, metabolic, and physiological factors affecting relative levels of acidogenesis and solventogenesis during anaerobic fermentation were investigated. In particular, CPC-PrOH3, a plasmid-free propanogenic E. coli strain derived by activating the Sbm operon on the genome, showed high levels of solventogenesis accounting for up to 85 % of dissimilated carbon. Anaerobic fed-batch cultivation of CPC-PrOH3 with glycerol as the major carbon source produced high titers of nearly 7 g/L 1-propanol and 31 g/L ethanol, implying its potential industrial applicability. The activated Sbm pathway served as an ancillary channel for consuming reducing equivalents upon anaerobic dissimilation of glycerol, resulting in an enhanced glycerol dissimilation and a major metabolic shift from acidogenesis to solventogenesis.

Clostridium pasteurianum is emerging as a prospective host for the production of biofuels and chemicals, and has recently been shown to directly consume electric current. Despite this growing biotechnological appeal, the organism’s genetics and central metabolism remain poorly understood. Here we present a concurrent genome sequence for the C. pasteurianum type strain and provide extensive genomic analysis of the organism’s defence mechanisms and central fermentative metabolism. Next generation genome sequencing produced reads corresponding to spontaneous excision of a novel phage, designated φ6013, which could be induced using mitomycin C and detected using PCR and transmission electron microscopy. Methylome analysis of sequencing reads provided a near-complete glimpse into the organism’s restriction-modification systems. We also unveiled the chief C. pasteurianum Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) locus, which was found to exemplify a Type I-B system. Finally, we show that C. pasteurianum possesses a highly complex fermentative metabolism whereby the metabolic pathways enlisted by the cell is governed by the degree of reductance of the substrate. Four distinct fermentation profiles, ranging from exclusively acidogenic to predominantly alcohologenic, were observed through redox consideration of the substrate. A detailed discussion of the organism’s central metabolism within the context of metabolic engineering is provided.

Doc number: 163 Abstract Background: Clostridium pasteurianum is one of the most promising biofuel producers within the genus Clostridium owing to its unique metabolic ability to ferment glycerol into butanol. Although an efficient means is available for introducing foreign DNA to C. pasteurianum , major genetic tools, such as gene knockout, knockdown, or genome editing, are lacking, preventing metabolic engineering of C. pasteurianum . Results: Here we present a methodology for performing chromosomal gene disruption in C. pasteurianum using the programmable lactococcus Ll.ltrB group II intron. Gene disruption was initially found to be impeded by inefficient electrotransformation of Escherichia coli -C. pasteurianum shuttle vectors, presumably due to host restriction. By assessing the ability of various vector deletion derivatives to electrotransform C. pasteurianum and probing the microorganism's methylome using next-generation sequence data, we identified a new C. pasteurianum Type I restriction-methylation system, CpaAII, with a predicted recognition sequence of 5[variant prime]-AAGNNNNNCTCC-3[variant prime] (N = A, C, G, or T). Following rescue of high-level electrotransformation via mutation of the sole CpaAII site within the shuttle vectors, we retargeted the intron to the cpaAIR gene encoding the CpaAI Type II restriction endonuclease (recognition site of 5[variant prime]-CGCG-3[variant prime]). Intron insertion was potentially hindered by low retrohoming efficiency, yet this limitation could be overcome by a procedure for enrichment of the intron insertion. The resulting ΔcpaAIR mutant strain was efficiently electrotransformed with M.FnuDII-unmethylated plasmid DNA. Conclusions: The markerless and plasmidless ΔcpaAIR mutant strain of C. pasteurianum developed in this study can serve as a general host strain for future genetic and metabolic manipulation. Further, the associated gene disruption protocol should not only serve as a guide for chromosomal gene inactivation studies involving mobile group II introns, but also prove invaluable for applying metabolic engineering strategies to C. pasteurianum .

Clostridium pasteurianum is receiving growing attention for its unique metabolic properties, particularly its ability to convert waste glycerol and glycerol-rich byproducts into butanol, a prospective biofuel. Genetic tool development and whole genome sequencing have recently been investigated to advance the genetic tractability of this potential industrial host. Nevertheless, methodologies for tuning gene expression through plasmid-borne expression and chromosomal gene downregulation are still absent. Here we demonstrate plasmid-borne heterologous gene expression and gene knockdown using antisense RNA in C. pasteurianum. We first employed a common thermophilic β-galactosidase (lacZ) gene reporter system from Thermoanaerobacterium thermosulfurogenes to characterize two promoters involved in the central fermentative metabolism of C. pasteurianum. Due to a higher level of constitutive lacZ expression compared to the ferredoxin gene (fdx) promoter, the thiolase (thl) promoter was selected to drive expression of asRNA. Expression of a lacZ asRNA resulted in 52%–58% downregulation of β-galactosidase activity compared to the control strain throughout the duration of culture growth. Subsequent implementation of our asRNA approach for downregulation of the native hydrogenase I gene (hydA) in C. pasteurianum resulted in altered end product distribution, characterized by an increase in production of reduced metabolites, particularly butyrate (40% increase) and ethanol (25% increase). Knockdown of hydA was also accompanied by increased acetate formation and lower levels of 1,3-propanediol, signifying a dramatic shift in cellular metabolism in response to inhibition of the hydrogenase enzyme. The methodologies described herein for plasmid-based heterologous gene expression and antisense-RNA-mediated gene knockdown should promote rational metabolic engineering of C. pasteurianum for enhanced production of butanol as a prospective biofuel.

The tetrahydroisoquinoline (THIQ) moiety is a privileged substructure of many bioactive natural products and semi-synthetic analogues. The plant kingdom manufactures more than 3,000 THIQ alkaloids, including the opioids morphine and codeine. While microbial species have been engineered to synthesize a few compounds from the benzylisoquinoline alkaloid (BIA) family of THIQs, low product titers impede industrial viability and limit access to the full chemical space. Here we report a THIQ platform by increasing yeast production of the central BIA intermediate (S)-reticuline to more than 3 g L-1, a 38,000-fold improvement over our first-generation strain. Gains in BIA output coincided with the formation of several substituted THIQs derived from host amino acid catabolism. Enabled by this activity, we repurposed the yeast Ehrlich pathway and demonstrate the synthesis of an array of unnatural THIQ scaffolds. This work provides a blueprint for synthesizing new privileged structures and will enable the targeted overproduction of thousands of THIQ products, including natural and semi-synthetic opioids.