Metabolic engineering of low-pH-tolerant non-model yeast, Issatchenkia orientalis, for production of citramalate

Zong-Yen Wu; Wan Sun; Yihui Shen; Jimmy Pratas; Patrick F. Suthers; Ping-Hung Hsieh; Sudharsan Dwaraknath; Joshua D. Rabinowitz; Costas D. Maranas; Zengyi Shao; Yasuo Yoshikuni

Metabolic Engineering Communications (Jun 2023)

Metabolic engineering of low-pH-tolerant non-model yeast, Issatchenkia orientalis, for production of citramalate

Zong-Yen Wu,
Wan Sun,
Yihui Shen,
Jimmy Pratas,
Patrick F. Suthers,
Ping-Hung Hsieh,
Sudharsan Dwaraknath,
Joshua D. Rabinowitz,
Costas D. Maranas,
Zengyi Shao,
Yasuo Yoshikuni

Affiliations

Zong-Yen Wu: Environmental Genomics and Systems Biology Division, Lawrence Berkeley National Laboratory, Berkeley, CA, 94720, USA
Wan Sun: Interdepartmental Microbiology Program, Iowa State University, Ames, IA, 50011-1027, USA; DOE Center for Advanced Bioenergy and Bioproducts Innovation, University of Illinois at Urbana-Champaign, Urbana, IL, 61801, USA
Yihui Shen: Department of Chemistry, Princeton University, Princeton, NJ, 08544, USA; Lewis Sigler Institute for Integrative Genomics, Princeton University, Princeton, NJ, 08540, USA
Jimmy Pratas: Department of Chemistry, Princeton University, Princeton, NJ, 08544, USA; Lewis Sigler Institute for Integrative Genomics, Princeton University, Princeton, NJ, 08540, USA
Patrick F. Suthers: Department of Chemical Engineering, The Pennsylvania State University, University Park, PA, 16802, USA; Center for Advanced Bioenergy and Bioproducts Innovation, The Pennsylvania State University, University Park, PA, 16802, USA
Ping-Hung Hsieh: US Department of Energy Joint Genome Institute, Lawrence Berkeley National Laboratory, Berkeley, CA, 94720, USA
Sudharsan Dwaraknath: US Department of Energy Joint Genome Institute, Lawrence Berkeley National Laboratory, Berkeley, CA, 94720, USA
Joshua D. Rabinowitz: Department of Chemistry, Princeton University, Princeton, NJ, 08544, USA; Lewis Sigler Institute for Integrative Genomics, Princeton University, Princeton, NJ, 08540, USA
Costas D. Maranas: Department of Chemical Engineering, The Pennsylvania State University, University Park, PA, 16802, USA; Center for Advanced Bioenergy and Bioproducts Innovation, The Pennsylvania State University, University Park, PA, 16802, USA
Zengyi Shao: Interdepartmental Microbiology Program, Iowa State University, Ames, IA, 50011-1027, USA; Department of Chemical and Biological Engineering, Iowa State University, Ames, IA, 50011, USA; NSF Engineering Research Center for Biorenewable Chemicals, Iowa State University, Ames, IA, 50011, USA; Bioeconomy Institute, Iowa State University, Ames, IA, 50011, USA; The Ames Laboratory, Ames, IA, 50011, USA; DOE Center for Advanced Bioenergy and Bioproducts Innovation, University of Illinois at Urbana-Champaign, Urbana, IL, 61801, USA; Corresponding author. Interdepartmental Microbiology Program, Iowa State University, Ames, IA, 50011-1027, USA.
Yasuo Yoshikuni: Environmental Genomics and Systems Biology Division, Lawrence Berkeley National Laboratory, Berkeley, CA, 94720, USA; Biological Systems and Engineering Division, Lawrence Berkeley National Laboratory, Berkeley, CA, 94720, USA; Center for Advanced Bioenergy and Bioproducts Innovation, Lawrence Berkeley National Laboratory, Berkeley, CA, 94720, USA; Global Center for Food, Land, and Water Resources, Hokkaido University, Hokkaido, 060-8589, Japan; Corresponding author. Environmental Genomics and Systems Biology Division, Lawrence Berkeley National Laboratory, Berkeley, CA, 94720, USA.

Journal volume & issue: Vol. 16
p. e00220

Abstract

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Methyl methacrylate (MMA) is an important petrochemical with many applications. However, its manufacture has a large environmental footprint. Combined biological and chemical synthesis (semisynthesis) may be a promising alternative to reduce both cost and environmental impact, but strains that can produce the MMA precursor (citramalate) at low pH are required. A non-conventional yeast, Issatchenkia orientalis, may prove ideal, as it can survive extremely low pH. Here, we demonstrate the engineering of I. orientalis for citramalate production. Using sequence similarity network analysis and subsequent DNA synthesis, we selected a more active citramalate synthase gene (cimA) variant for expression in I. orientalis. We then adapted a piggyBac transposon system for I. orientalis that allowed us to simultaneously explore the effects of different cimA gene copy numbers and integration locations. A batch fermentation showed the genome-integrated-cimA strains produced 2.0 g/L citramalate in 48 h and a yield of up to 7% mol citramalate/mol consumed glucose. These results demonstrate the potential of I. orientalis as a chassis for citramalate production.

Published in Metabolic Engineering Communications

ISSN: 2214-0301 (Online)
Publisher: Elsevier
Country of publisher: Netherlands
LCC subjects: Technology: Chemical technology: Biotechnology; Science: Biology (General)
Website: http://www.journals.elsevier.com/metabolic-engineering-communications/

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