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Title: OptSSeq: High-Throughput Sequencing Readout of Growth Enrichment Defines Optimal Gene Expression Elements for Homoethanologenesis

Abstract

The optimization of synthetic pathways is a central challenge in metabolic engineering. OptSSeq (Optimization by Selection and Sequencing) is one approach to this challenge. OptSSeq couples selection of optimal enzyme expression levels linked to cell growth rate with high-throughput sequencing to track enrichment of gene expression elements (promoters and ribosomebinding sites) from a combinatorial library. OptSSeq yields information on both optimal and suboptimal enzyme levels, and helps identify constraints that limit maximal product formation. Here we report a proof-of-concept implementation of OptSSeq using homoethanologenesis, a two-step pathway consisting of pyruvate decarboxylase (Pdc) and alcohol dehydrogenase (Adh) that converts pyruvate to ethanol and is naturally optimized in the bacterium Zymomonas mobilis. We used OptSSeq to determine optimal gene expression elements and enzyme levels for Z. mobilis Pdc, AdhA, and AdhB expressed in Escherichia coli. By varying both expression signals and gene order, we identified an optimal solution using only Pdc and AdhB. We resolved current uncertainty about the functions of the Fe2+-dependent AdhB and Zn2+- dependent AdhA by showing that AdhB is preferred over AdhA for rapid growth in both E. coli and Z. mobilis. Finally, by comparing predictions of growth-linked metabolic flux to enzyme synthesis costs, we established that optimalmore » E. coli homoethanologenesis was achieved by our best pdc-adhB expression cassette and that the remaining constraints lie in the E. coli metabolic network or inefficient Pdc or AdhB function in E. coli. Furthermore, OptSSeq is a general tool for synthetic biology to tune enzyme levels in any pathway whose optimal function can be linked to cell growth or survival.« less

Authors:
 [1];  [1]
  1. DOE Great Lakes Bioenergy Research Center, University of Wisconsin—Madison, Madison, Wisconsin 53726, United States
Publication Date:
Research Org.:
Univ. of Wisconsin, Madison, WI (United States)
Sponsoring Org.:
USDOE Office of Science (SC), Biological and Environmental Research (BER)
OSTI Identifier:
1294691
Alternate Identifier(s):
OSTI ID: 1337746
Grant/Contract Number:  
FC02-07ER64494
Resource Type:
Published Article
Journal Name:
ACS Synthetic Biology
Additional Journal Information:
Journal Name: ACS Synthetic Biology Journal Volume: 5 Journal Issue: 12; Journal ID: ISSN 2161-5063
Publisher:
American Chemical Society
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES; 37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CHEMISTRY; combinatorial optimization; ethanol; metabolic engineering; promoter; ribosome binding site; synthetic biology

Citation Formats

Ghosh, Indro Neil, and Landick, Robert. OptSSeq: High-Throughput Sequencing Readout of Growth Enrichment Defines Optimal Gene Expression Elements for Homoethanologenesis. United States: N. p., 2016. Web. doi:10.1021/acssynbio.6b00121.
Ghosh, Indro Neil, & Landick, Robert. OptSSeq: High-Throughput Sequencing Readout of Growth Enrichment Defines Optimal Gene Expression Elements for Homoethanologenesis. United States. https://doi.org/10.1021/acssynbio.6b00121
Ghosh, Indro Neil, and Landick, Robert. Mon . "OptSSeq: High-Throughput Sequencing Readout of Growth Enrichment Defines Optimal Gene Expression Elements for Homoethanologenesis". United States. https://doi.org/10.1021/acssynbio.6b00121.
@article{osti_1294691,
title = {OptSSeq: High-Throughput Sequencing Readout of Growth Enrichment Defines Optimal Gene Expression Elements for Homoethanologenesis},
author = {Ghosh, Indro Neil and Landick, Robert},
abstractNote = {The optimization of synthetic pathways is a central challenge in metabolic engineering. OptSSeq (Optimization by Selection and Sequencing) is one approach to this challenge. OptSSeq couples selection of optimal enzyme expression levels linked to cell growth rate with high-throughput sequencing to track enrichment of gene expression elements (promoters and ribosomebinding sites) from a combinatorial library. OptSSeq yields information on both optimal and suboptimal enzyme levels, and helps identify constraints that limit maximal product formation. Here we report a proof-of-concept implementation of OptSSeq using homoethanologenesis, a two-step pathway consisting of pyruvate decarboxylase (Pdc) and alcohol dehydrogenase (Adh) that converts pyruvate to ethanol and is naturally optimized in the bacterium Zymomonas mobilis. We used OptSSeq to determine optimal gene expression elements and enzyme levels for Z. mobilis Pdc, AdhA, and AdhB expressed in Escherichia coli. By varying both expression signals and gene order, we identified an optimal solution using only Pdc and AdhB. We resolved current uncertainty about the functions of the Fe2+-dependent AdhB and Zn2+- dependent AdhA by showing that AdhB is preferred over AdhA for rapid growth in both E. coli and Z. mobilis. Finally, by comparing predictions of growth-linked metabolic flux to enzyme synthesis costs, we established that optimal E. coli homoethanologenesis was achieved by our best pdc-adhB expression cassette and that the remaining constraints lie in the E. coli metabolic network or inefficient Pdc or AdhB function in E. coli. Furthermore, OptSSeq is a general tool for synthetic biology to tune enzyme levels in any pathway whose optimal function can be linked to cell growth or survival.},
doi = {10.1021/acssynbio.6b00121},
journal = {ACS Synthetic Biology},
number = 12,
volume = 5,
place = {United States},
year = {2016},
month = {8}
}

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