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

Ghosh, Indro Neil; Landick, Robert

doi:10.1021/acssynbio.6b00121

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

Journal Article · Mon Aug 01 00:00:00 EDT 2016 · ACS Synthetic Biology

DOI:https://doi.org/10.1021/acssynbio.6b00121· OSTI ID:1294691

Ghosh, Indro Neil ^[1]; Landick, Robert ^[1]

DOE Great Lakes Bioenergy Research Center, University of Wisconsin—Madison, Madison, Wisconsin 53726, United States

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.

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Research Organization:: Univ. of Wisconsin, Madison, WI (United States)

Sponsoring Organization:: USDOE Office of Science (SC), Biological and Environmental Research (BER)

Grant/Contract Number:: FC02-07ER64494

OSTI ID:: 1294691

Alternate ID(s):: OSTI ID: 1337746

Journal Information:: ACS Synthetic Biology, Journal Name: ACS Synthetic Biology Vol. 5 Journal Issue: 12; ISSN 2161-5063

Publisher:: American Chemical SocietyCopyright Statement

Country of Publication:: United States

Language:: English

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Cited by: 11 works

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59 BASIC BIOLOGICAL SCIENCES
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ribosome binding site
synthetic biology

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

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