skip to main content
US FlagAn official website of the United States government
dot gov icon
Official websites use .gov
A .gov website belongs to an official government organization in the United States.
https lock icon
Secure .gov websites use HTTPS
A lock ( lock ) or https:// means you've safely connected to the .gov website. Share sensitive information only on official, secure websites.

Explore Research Products in the PAR
It may take a few hours for recently added research products to appear in PAR search results.


Title: Engineering controllable alteration of malonyl-CoA levels to enhance polyketide production
Abstract Heterologous expression of polyketide synthase (PKS) genes inEscherichia colihas enabled the production of various valuable natural and synthetic products. However, the limited availability of malonyl-CoA (M-CoA) inE. coliremains a substantial impediment to high-titer polyketide production. Here we address this limitation by disrupting the native M-CoA biosynthetic pathway and introducing an orthogonal pathway comprising a malonate transporter and M-CoA ligase, enabling efficient M-CoA biosynthesis under malonate supplementation. This approach substantially increases M-CoA levels, enhancing fatty acid and polyketide titers while reducing the promiscuous activity of PKSs toward undesired acyl-CoA substrates. Subsequent adaptive laboratory evolution of these strains provides insights into M-CoA regulation and identifies mutations that further boost M-CoA and polyketide production. This strategy improvesE. colias a host for polyketide biosynthesis and advances understanding of M-CoA metabolism in microbial systems.  more » « less
Award ID(s):
2036849
PAR ID:
10637344
Author(s) / Creator(s):
; ; ; ; ; ; ; ; ; ;
Publisher / Repository:
Nature Chemical Biology
Date Published:
Journal Name:
Nature Chemical Biology
Volume:
21
Issue:
8
ISSN:
1552-4450
Page Range / eLocation ID:
1214 to 1225
Format(s):
Medium: X
Sponsoring Org:
National Science Foundation
More Like this
  1. ; ; ; (, mSphere)
    Bradford, Patricia A. (Ed.)
    ABSTRACT Efflux and motility are two key biological functions in bacteria. Recent findings have shown that efflux impacts flagellum biosynthesis and motility inEscherichia coliand other bacteria. AcrR is known to be the major transcriptional repressor of AcrAB-TolC, the main multidrug efflux pump inE. coliand otherEnterobacteriaceae. However, the underlying molecular mechanisms of how efflux and motility are co-regulated remain poorly understood. Here, we have studied the role of AcrR in direct regulation of motility inE. coli. By combining bioinformatics, electrophoretic mobility shift assays (EMSAs), gene expression, and motility experiments, we have found that AcrR represses motility inE. coliby directly repressing transcription of theflhDCoperon, but not the other flagellum genes/operons tested.flhDCencodes the master regulator of flagellum biosynthesis and motility genes. We found that such regulation primarily occurs by direct binding of AcrR to theflhDCpromoter region containing the first of the two predicted AcrR-binding sites identified in this promoter. This is the first report of direct regulation by AcrR of genes unrelated to efflux or detoxification. Moreover, we report that overexpression of AcrR restores to parental levels the increased swimming motility previously observed inE. colistrains without a functional AcrAB-TolC pump, and that such effect by AcrR is prevented by the AcrR ligand and AcrAB-TolC substrate ethidium bromide. Based on these and prior findings, we provide a novel model in which AcrR senses efflux and then co-regulates efflux and motility inE. colito maintain homeostasis and escape hazards. IMPORTANCEEfflux and motility play a major role in bacterial growth, colonization, and survival. InEscherichia coli, the transcriptional repressor AcrR is known to directly repress efflux and was later found to also repress flagellum biosynthesis and motility by Kim et al. (J Microbiol Biotechnol 26:1824–1828, 2016, doi: 10.4014/jmb.1607.07058). However, it remained unknown whether AcrR represses flagellum biosynthesis and motility directly and through which target genes, or indirectly because of altering the amount of efflux. This study reveals that AcrR represses flagellum biosynthesis and motility by directly repressing the expression of theflhDCmaster regulator of flagellum biosynthesis and motility genes, but not the other flagellum genes tested. We also show that the antimicrobial, efflux pump substrate, and AcrR ligand ethidium bromide regulates motility via AcrR. Overall, these findings support a novel model of direct co-regulation of efflux and motility mediated by AcrR in response to stress inE. coli. 
    more » « less
  2. ; ; ; ; ; ; ; ; ; ; et al (, Nature Communications)
    Abstract Fatty acid desaturation is central to metazoan lipid metabolism and provides building blocks of membrane lipids and precursors of diverse signaling molecules. Nutritional conditions and associated microbiota regulate desaturase expression, but the underlying mechanisms have remained unclear. Here, we show that endogenous and microbiota-dependent small molecule signals promote lipid desaturation via the nuclear receptor NHR-49/PPARα inC. elegans. Untargeted metabolomics of a β-oxidation mutant,acdh-11, in which expression of the stearoyl-CoA desaturase FAT-7/SCD1 is constitutively increased, revealed accumulation of a β-cyclopropyl fatty acid, becyp#1, that potently activatesfat-7expression via NHR-49. Biosynthesis of becyp#1 is strictly dependent on expression of cyclopropane synthase by associated bacteria, e.g.,E. coli. Screening for structurally related endogenous metabolites revealed a β-methyl fatty acid, bemeth#1, which mimics the activity of microbiota-dependent becyp#1 but is derived from a methyltransferase,fcmt-1, that is conserved across Nematoda and likely originates from bacterial cyclopropane synthase via ancient horizontal gene transfer. Activation offat-7expression by these structurally similar metabolites is controlled by distinct mechanisms, as microbiota-dependent becyp#1 is metabolized by a dedicated β-oxidation pathway, while the endogenous bemeth#1 is metabolized via α-oxidation. Collectively, we demonstrate that evolutionarily related biosynthetic pathways in metazoan host and associated microbiota converge on NHR-49/PPARα to regulate fat desaturation. 
    more » « less
  3. ; ; ; ; ; (, Microbial Cell Factories)
    null (Ed.)
    Abstract Background Noncanonical redox cofactors are emerging as important tools in cell-free biosynthesis to increase the economic viability, to enable exquisite control, and to expand the range of chemistries accessible. However, these noncanonical redox cofactors need to be biologically synthesized to achieve full integration with renewable biomanufacturing processes. Results In this work, we engineered Escherichia coli cells to biosynthesize the noncanonical cofactor nicotinamide mononucleotide (NMN + ), which has been efficiently used in cell-free biosynthesis. First, we developed a growth-based screening platform to identify effective NMN + biosynthetic pathways in E. coli . Second, we explored various pathway combinations and host gene disruption to achieve an intracellular level of ~ 1.5 mM NMN + , a 130-fold increase over the cell’s basal level, in the best strain, which features a previously uncharacterized nicotinamide phosphoribosyltransferase (NadV) from Ralstonia solanacearum. Last, we revealed mechanisms through which NMN + accumulation impacts E. coli cell fitness, which sheds light on future work aiming to improve the production of this noncanonical redox cofactor. Conclusion These results further the understanding of effective production and integration of NMN + into E. coli . This may enable the implementation of NMN + -directed biocatalysis without the need for exogenous cofactor supply. 
    more » « less
  4. ; ; ; (, Frontiers in Microbiology)
    IntroductionThe rise in extended-spectrum beta-lactamase (ESBL)-producingEnterobacteriaceaein dairy cattle farms poses a risk to human health as they can spread to humans through the food chain, including raw milk. This study was designed to determine the status, antimicrobial resistance, and pathogenic potential of ESBL-producing -E. coliand -Klebsiellaspp. isolates from bulk tank milk (BTM). MethodsThirty-three BTM samples were collected from 17 dairy farms and screened for ESBL-E. coliand -Klebsiellaspp. on CHROMagar ESBL plates. All isolates were confirmed by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) and subjected to antimicrobial susceptibility testing and whole genome sequencing (WGS). ResultsTen presumptive ESBL-producing bacteria, eightE. coli, and twoK. pneumoniaewere isolated. The prevalence of ESBL-E. coliand -K. pneumoniaein BTM was 21.2% and 6.1%, respectively. ESBL-E. coliwere detected in 41.2% of the study farms. Seven of the ESBL-E. coliisolates were multidrug resistant (MDR). The two ESBL-producingK. pneumoniaeisolates were resistant to ceftriaxone. Seven ESBL-E. colistrains carry theblaCTX-Mgene, and five of them co-harboredblaTEM-1. ESBL-E. colico-harboredblaCTX-Mwith other resistance genes, includingqnrB19,tet(A),aadA1,aph(3’’)-Ib,aph(6)-Id),floR,sul2, and chromosomal mutations (gyrA, gyrB, parC, parE, and pmrB). MostE. coliresistance genes were associated with mobile genetic elements, mainly plasmids. Six sequence types (STs) ofE. coliwere detected. All ESBL-E. coliwere predicted to be pathogenic to humans. Four STs (three ST10 and ST69) were high-risk clones ofE. coli. Up to 40 virulence markers were detected in allE. coliisolates. One of theK. pneumoniaewas ST867; the other was novel strain.K. pneumoniaeisolates carried three types of beta-lactamase genes (blaCTX-M,blaTEM-1andblaSHV). The novelK. pneumoniaeST also carried a novel IncFII(K) plasmid ST. ConclusionDetection of high-risk clones of MDR ESBL-E. coliand ESBL-K. pneumoniaein BTM indicates that raw milk could be a reservoir of potentially zoonotic ESBL-E. coliand -K. pneumoniae. 
    more » « less
  5. ; ; ; ; ; (, Journal of Industrial Microbiology and Biotechnology)
    Abstract Mevalonate is a key precursor in isoprenoid biosynthesis and a promising commodity chemical. Although mevalonate is a native metabolite in Saccharomyces cerevisiae, its production is challenged by the relatively low flux toward acetyl-CoA in this yeast. In this study we explore different approaches to increase acetyl-CoA supply in S. cerevisiae to boost mevalonate production. Stable integration of a feedback-insensitive acetyl-CoA synthetase (Se-acsL641P) from Salmonella enterica and the mevalonate pathway from Enterococcus faecalis results in the production of 1,390 ± 10 mg/l of mevalonate from glucose. While bifid shunt enzymes failed to improve titers in high-producing strains, inhibition of squalene synthase (ERG9) results in a significant enhancement. Finally, increasing coenzyme A (CoA) biosynthesis by overexpression of pantothenate kinase (CAB1) and pantothenate supplementation further increased production to 3,830 ± 120 mg/l. Using strains that combine these strategies in lab-scale bioreactors results in the production of 13.3 ± 0.5 g/l, which is ∼360-fold higher than previously reported mevalonate titers in yeast. This study demonstrates the feasibility of engineering S. cerevisiae for high-level mevalonate production. 
    more » « less
  • Citation Formats
  • MLA
  • APA
  • Chicago
  • BibTeX
  • Text Encoded Conversion
  • XML
  • Save / Share this Record
  • Send to Email