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Although this carbon stream provides a rich source of sugars, other organic compounds, such as acetate, can be used by microbial hosts. Here, we examined the growth and isoprenol production in a\n                      <jats:italic>Pseudomonas putida<\/jats:italic>\n                      strain pre-tolerized (\u201cPT\u201d) background where its native isoprenol catabolism pathway is deleted, using glucose and acetate as carbon sources. We found that PT displays impaired growth in minimal medium containing acetate and often fails to grow in glucose-acetate medium. Using a mutant recovery-based approach, we generated tolerized strains that overcame these limitations, achieving fast growth and isoprenol production in the mixed carbon feed. Changes in the glucose and acetate assimilation routes, including an upregulation in PP_0154 (SpcC, succinyl-CoA:acetate CoA-transferase) and differential expression of the gluconate assimilation pathways, were key for higher isoprenol titers in the tolerized strains, whereas a different set of mechanisms were likely enabling tolerance phenotypes in media containing acetate. Among these, a coproporphyrinogen-III oxidase (HemN) was upregulated across all tolerized strains and in one isolate required for acetate tolerance. Utilizing a defined glucose and acetate mixture ratio reflective of lignocellulosic feedstocks for isoprenol production in\n                      <jats:italic>P. putida<\/jats:italic>\n                      allowed us to obtain insights into the dynamics and challenges unique to dual carbon source utilization that are obscured when studied separately. Together, this enabled the development of a\n                      <jats:italic>P. putida<\/jats:italic>\n                      bioconversion chassis able to use a more complex carbon stream to produce isoprenol.\n                    <\/jats:p>\n                    <jats:sec>\n                      <jats:title>IMPORTANCE<\/jats:title>\n                      <jats:p>\n                        Acetate is a relatively abundant component of many lignocellulosic carbon streams and has the potential to be used together with sugars, especially in microbes with versatile catabolism such as\n                        <jats:italic>P. putida<\/jats:italic>\n                        . However, the use of mixed carbon streams necessitates additional optimization. Furthermore, the use of\n                        <jats:italic>P. putida<\/jats:italic>\n                        for the production of the biofuel target, isoprenol, requires the use of engineered strains that have additional growth and production constraints when cultivated in acetate and glucose mixtures. In this study, we generate acetate-tolerant\n                        <jats:italic>P. putida<\/jats:italic>\n                        strains that overcome these challenges and examine their ability to produce isoprenol. We show that acetate tolerance and isoprenol production, although independent phenotypes, can both be optimized in a given\n                        <jats:italic>P. putida<\/jats:italic>\n                        strain. Using proteomics and whole genome sequencing, we examine the molecular basis of both phenotypes and show that tolerance to acetate can occur via alternate routes and result in different impacts on isoprenol production.\n                      <\/jats:p>\n                    <\/jats:sec>\n                  <\/jats:sec>","DOI":"10.1128\/aem.02123-24","type":"journal-article","created":{"date-parts":[[2025,3,20]],"date-time":"2025-03-20T08:43:41Z","timestamp":1742460221000},"update-policy":"https:\/\/doi.org\/10.1128\/asmj-crossmark-policy-page","source":"Crossref","is-referenced-by-count":4,"title":["Alternate routes to acetate tolerance lead to varied isoprenol production from mixed carbon sources in\n                    <i>Pseudomonas putida<\/i>"],"prefix":"10.1128","volume":"91","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-3645-6363","authenticated-orcid":false,"given":"Guilherme M. V.","family":"de Siqueira","sequence":"first","affiliation":[{"name":"The Joint BioEnergy Institute, Lawrence Berkeley National Laboratory","place":["Emeryville, USA"]},{"name":"Biological Systems and Engineering Division, Lawrence Berkeley National Laboratory","place":["Berkeley, USA"]},{"name":"Department of Biology, Faculty of Philosophy, Sciences and Letters at Ribeir\u00e3o Preto, University of S\u00e3o Paulo","place":["Ribeir\u00e3o Preto, Brazil"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Aparajitha","family":"Srinivasan","sequence":"additional","affiliation":[{"name":"The Joint BioEnergy Institute, Lawrence Berkeley National Laboratory","place":["Emeryville, USA"]},{"name":"Biological Systems and Engineering Division, Lawrence Berkeley National Laboratory","place":["Berkeley, USA"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yan","family":"Chen","sequence":"additional","affiliation":[{"name":"The Joint BioEnergy Institute, Lawrence Berkeley National Laboratory","place":["Emeryville, USA"]},{"name":"Biological Systems and Engineering Division, Lawrence Berkeley National Laboratory","place":["Berkeley, USA"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jennifer W.","family":"Gin","sequence":"additional","affiliation":[{"name":"The Joint BioEnergy Institute, Lawrence Berkeley National Laboratory","place":["Emeryville, USA"]},{"name":"Biological Systems and Engineering Division, Lawrence Berkeley National Laboratory","place":["Berkeley, USA"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Christopher J.","family":"Petzold","sequence":"additional","affiliation":[{"name":"The Joint BioEnergy Institute, Lawrence Berkeley National Laboratory","place":["Emeryville, USA"]},{"name":"Biological Systems and Engineering Division, Lawrence Berkeley National Laboratory","place":["Berkeley, USA"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Taek Soon","family":"Lee","sequence":"additional","affiliation":[{"name":"The Joint BioEnergy Institute, Lawrence Berkeley National Laboratory","place":["Emeryville, USA"]},{"name":"Biological Systems and Engineering Division, Lawrence Berkeley National Laboratory","place":["Berkeley, USA"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Mar\u00eda-Eugenia","family":"Guazzaroni","sequence":"additional","affiliation":[{"name":"Department of Biology, Faculty of Philosophy, Sciences and Letters at Ribeir\u00e3o Preto, University of S\u00e3o Paulo","place":["Ribeir\u00e3o Preto, Brazil"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-4974-3863","authenticated-orcid":false,"given":"Thomas","family":"Eng","sequence":"additional","affiliation":[{"name":"The Joint BioEnergy Institute, Lawrence Berkeley National Laboratory","place":["Emeryville, USA"]},{"name":"Biological Systems and Engineering Division, Lawrence Berkeley National Laboratory","place":["Berkeley, USA"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-6513-7425","authenticated-orcid":true,"given":"Aindrila","family":"Mukhopadhyay","sequence":"additional","affiliation":[{"name":"The Joint BioEnergy Institute, Lawrence Berkeley National Laboratory","place":["Emeryville, USA"]},{"name":"Biological Systems and Engineering Division, Lawrence Berkeley National Laboratory","place":["Berkeley, USA"]},{"name":"Environmental Genomics and Systems Biology Division, Lawrence Berkeley National Laboratory","place":["Berkeley, USA"]}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"235","reference":[{"key":"e_1_3_4_2_2","unstructured":"Langholtz M. 2024. 2023 billion-ton report. 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