scholarly journals Anaerobic Fungal Mevalonate Pathway Genomic Biases Lead to Heterologous Toxicity Underpredicted by Codon Adaptation Indices

2021 ◽  
Vol 9 (9) ◽  
pp. 1986
Author(s):  
Ethan T. Hillman ◽  
Elizabeth M. Frazier ◽  
Evan K. Shank ◽  
Adrian N. Ortiz-Velez ◽  
Jacob A. Englaender ◽  
...  
Keyword(s):  
Natural Product ◽  
Mevalonate Pathway ◽  
Codon Optimization ◽  
Biosynthetic Pathways ◽  
Anaerobic Fungi ◽  
Genome Composition ◽  
E Coli ◽  
Growth Defects ◽  
Fungal Enzyme ◽  
Limited Success

Anaerobic fungi are emerging biotechnology platforms with genomes rich in biosynthetic potential. Yet, the heterologous expression of their biosynthetic pathways has had limited success in model hosts like E. coli. We find one reason for this is that the genome composition of anaerobic fungi like P. indianae are extremely AT-biased with a particular preference for rare and semi-rare AT-rich tRNAs in E coli, which are not explicitly predicted by standard codon adaptation indices (CAI). Native P. indianae genes with these extreme biases create drastic growth defects in E. coli (up to 69% reduction in growth), which is not seen in genes from other organisms with similar CAIs. However, codon optimization rescues growth, allowing for gene evaluation. In this manner, we demonstrate that anaerobic fungal homologs such as PI.atoB are more active than S. cerevisiae homologs in a hybrid pathway, increasing the production of mevalonate up to 2.5 g/L (more than two-fold) and reducing waste carbon to acetate by ~90% under the conditions tested. This work demonstrates the bioproduction potential of anaerobic fungal enzyme homologs and how the analysis of codon utilization enables the study of otherwise difficult to express genes that have applications in biocatalysis and natural product discovery.

scholarly journals Computational Tools for Discovering and Engineering Natural Product Biosynthetic Pathways

iScience ◽  
2020 ◽  
Vol 23 (1) ◽  
pp. 100795
Author(s):  
Hengqian Ren ◽  
Chengyou Shi ◽  
Huimin Zhao
Keyword(s):  
Natural Product ◽  
Biosynthetic Pathways ◽  
Computational Tools

Increased Production of Recombinant O-Phospho-L-Serine Sulfhydrylase from the Hyperthermophilic Archaeon Aeropyrum pernix K1 Using Escherichia coli

2019 ◽  
Vol 8 (1) ◽  
pp. 15-23
Author(s):  
Takashi Nakamura ◽  
Emi Takeda ◽  
Tomoko Kiryu ◽  
Kentaro Mori ◽  
Miyu Ohori ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Amino Acids ◽  
Industrial Production ◽  
Codon Optimization ◽  
Scale Production ◽  
Enzymatic Production ◽  
Hyperthermophilic Archaeon ◽  
E Coli ◽  
Aeropyrum Pernix ◽  
Natural Amino Acids

Background: O-phospho-L-serine sulfhydrylase from the hyperthermophilic archaeon Aeropyrum pernix K1 (ApOPSS) is thermostable and tolerant to organic solvents. It can produce nonnatural amino acids in addition to L-cysteine. Objective: We aimed to obtain higher amounts of ApOPSS compared to those reported with previous methods for the convenience of research and for industrial production of L-cysteine and non-natural amino acids. Method: We performed codon optimization of cysO that encodes ApOPSS, for optimal expression in Escherichia coli. We then examined combinations of conditions such as the host strain, plasmid, culture medium, and isopropyl β-D-1-thiogalactopyranoside (IPTG) concentration to improve ApOPSS yield. Results and Discussion: E. coli strain Rosetta (DE3) harboring the expression plasmid pQE-80L with the codon-optimized cysO was cultured in Terrific broth with 0.01 mM IPTG at 37°C for 48 h to yield a 10-times higher amount of purified ApOPSS (650 mg·L-1) compared to that obtained by the conventional method (64 mg·L-1). We found that the optimal culture conditions along with codon optimization were essential for the increased ApOPSS production. The expressed ApOPSS had a 6-histidine tag at the N-terminal, which did not affect its activity. This method may facilitate the industrial production of cysteine and non-natural amino acids using ApOPSS. Conclusion: We conclude that these results could be used in applied research on enzymatic production of L-cysteine in E. coli, large scale production of non-natural amino acids, an enzymatic reaction in organic solvent, and environmental remediation by sulfur removal.

Cobalamin-dependent radicalS-adenosyl-l-methionine enzymes in natural product biosynthesis

2018 ◽  
Vol 35 (8) ◽  
pp. 707-720 ◽  
Author(s):  
Susan C. Wang
Keyword(s):  
Natural Product ◽  
Biosynthetic Pathways ◽  
Natural Product Biosynthesis

This highlight examines the functions of cobalamin-dependent radicalS-adenosyl-l-methionine enzymes that catalyse chemically-challenging reactions in several bacterial natural product biosynthetic pathways.

scholarly journals Modulating the Precursor and Terpene Synthase Supply for the Whole-Cell Biocatalytic Production of the Sesquiterpene (+)-Zizaene in a Pathway Engineered E. coli

Genes ◽  
2019 ◽  
Vol 10 (6) ◽  
pp. 478 ◽  
Author(s):  
Francisco Aguilar ◽  
Thomas Scheper ◽  
Sascha Beutel
Keyword(s):  
Essential Oil ◽  
Metabolic Flux ◽  
Mevalonate Pathway ◽  
Terpene Synthase ◽  
Methylerythritol Phosphate Pathway ◽  
Main Compound ◽  
E Coli ◽  
Methylerythritol Phosphate ◽  
Cosmetic Industry ◽  
Multiple Copies

The vetiver essential oil from Chrysopogon zizanioides contains fragrant sesquiterpenes used widely in the formulation of nearly 20% of men’s cosmetics. The growing demand and issues in the supply have raised interest in the microbial production of the sesquiterpene khusimol, the main compound of the vetiver essential oil due to its woody smell. In this study, we engineered the biosynthetic pathway for the production of (+)-zizaene, the immediate precursor of khusimol. A systematic approach of metabolic engineering in Escherichia coli was applied to modulate the critical bottlenecks of the metabolic flux towards (+)-zizaene. Initially, production of (+)-zizaene was possible with the endogenous methylerythritol phosphate pathway and the codon-optimized zizaene synthase (ZS). Raising the precursor E,E-farnesyl diphosphate supply through the mevalonate pathway improved the (+)-zizaene titers 2.7-fold, although a limitation of the ZS supply was observed. To increase the ZS supply, distinct promoters were tested for the expression of the ZS gene, which augmented 7.2-fold in the (+)-zizaene titers. Final metabolic enhancement for the ZS supply by using a multi-plasmid strain harboring multiple copies of the ZS gene improved the (+)-zizaene titers 1.3-fold. The optimization of the fermentation conditions increased the (+)-zizaene titers 2.2-fold, achieving the highest (+)-zizaene titer of 25.09 mg L−1. This study provides an alternative strategy to enhance the terpene synthase supply for the engineering of isoprenoids. Moreover, it demonstrates the development of a novel microbial platform for the sustainable production of fragrant molecules for the cosmetic industry.

scholarly journals Regulation of Antimycin Biosynthesis Is Controlled by the ClpXP Protease

mSphere ◽  
2020 ◽  
Vol 5 (2) ◽  
Author(s):  
Bohdan Bilyk ◽  
Sora Kim ◽  
Asif Fazal ◽  
Tania A. Baker ◽  
Ryan F. Seipke
Keyword(s):  
Natural Products ◽  
Rna Polymerase ◽  
Natural Product ◽  
Morphological Differentiation ◽  
Dynamic Responses ◽  
Biosynthetic Pathways ◽  
Recognition Sequence ◽  
Σ Factor

ABSTRACT The survival of any microbe relies on its ability to respond to environmental change. Use of extracytoplasmic function (ECF) RNA polymerase sigma (σ) factors is a major strategy enabling dynamic responses to extracellular signals. Streptomyces species harbor a large number of ECF σ factors, nearly all of which are uncharacterized, but those that have been characterized generally regulate genes required for morphological differentiation and/or response to environmental stress, except for σAntA, which regulates starter-unit biosynthesis in the production of antimycin, an anticancer compound. Unlike a canonical ECF σ factor, whose activity is regulated by a cognate anti-σ factor, σAntA is an orphan, raising intriguing questions about how its activity may be controlled. Here, we reconstituted in vitro ClpXP proteolysis of σAntA but not of a variant lacking a C-terminal di-alanine motif. Furthermore, we show that the abundance of σAntA in vivo was enhanced by removal of the ClpXP recognition sequence and that levels of the protein rose when cellular ClpXP protease activity was abolished. These data establish direct proteolysis as an alternative and, thus far, unique control strategy for an ECF RNA polymerase σ factor and expands the paradigmatic understanding of microbial signal transduction regulation. IMPORTANCE Natural products produced by Streptomyces species underpin many industrially and medically important compounds. However, the majority of the ∼30 biosynthetic pathways harbored by an average species are not expressed in the laboratory. This unrevealed biochemical diversity is believed to comprise an untapped resource for natural product drug discovery. Major roadblocks preventing the exploitation of unexpressed biosynthetic pathways are a lack of insight into their regulation and limited technology for activating their expression. Our findings reveal that the abundance of σAntA, which is the cluster-situated regulator of antimycin biosynthesis, is controlled by the ClpXP protease. These data link proteolysis to the regulation of natural product biosynthesis for the first time to our knowledge, and we anticipate that this will emerge as a major strategy by which actinobacteria regulate production of their natural products. Further study of this process will advance understanding of how expression of secondary metabolism is controlled and will aid pursuit of activating unexpressed biosynthetic pathways.

scholarly journals Toward Biosynthetic Design and Implementation of Escherichia coli-Derived Paclitaxel and Other Heterologous Polyisoprene Compounds

2012 ◽  
Vol 78 (8) ◽  
pp. 2497-2504 ◽  
Author(s):  
Ming Jiang ◽  
Gregory Stephanopoulos ◽  
Blaine A. Pfeifer
Keyword(s):  
Escherichia Coli ◽  
Natural Products ◽  
Natural Product ◽  
Cellular Metabolism ◽  
Compound Formation ◽  
Natural Product Biosynthesis ◽  
Content Type ◽  
E Coli ◽  
Design And Implementation ◽  
Final Compound

ABSTRACTEscherichia colioffers unparalleled engineering capacity in the context of heterologous natural product biosynthesis. However, as with other heterologous hosts, cellular metabolism must be designed or redesigned to support final compound formation. This task is at once complicated and aided by the fact that the cell does not natively produce an abundance of natural products. As a result, the metabolic engineer avoids complicated interactions with native pathways closely associated with the outcome of interest, but this convenience is tempered by the need to implement the required metabolism to allow functional biosynthesis. This review focuses on engineeringE. colifor the purpose of polyisoprene formation, as it is related to isoprenoid compounds currently being pursued through a heterologous approach. In particular, the review features the compound paclitaxel and early efforts to design and overproduce intermediates throughE. coli.

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