Cre-lox66/lox71-Based Elimination of Phosphotransacetylase or Acetaldehyde Dehydrogenase Shifted Carbon Flux in Acetogen Rendering Selective Overproduction of Ethanol or Acetate

Abstract

Acetogen strain Clostridium sp. MT1121 produced 300 mM acetate (p<0.005) and 321 mM ethanol (p<0.005) from synthesis gas (syngas) blend 60 % CO and 40 % H(2). Clostridium sp. MT1121 was metabolically engineered to eliminate production of either acetate or acetaldehyde during syngas fermentation. We used Cre-lox66/lox71-based gene removal system to eliminate either phosphotransacetylase (pta), or acetaldehyde dehydrogenase (aldh). The resulted biocatalyst with eliminated pta increased ethanol yield to 610 mM (p<0.005). Inactivation of pta rendered only 502 mM of ethanol (p<0.005). The acetogen biocatalyst with eliminated aldh produced 450 mM acetate (p<0.005). The role of cell energy pool preservation for re-directed carbon flux is discussed. This is the first report on time- and cost-efficient gene elimination in acetogens using lox66/lox71 gene elimination system.

Trash to treasure: production of biofuels and commodity chemicals via syngas fermenting microorganisms

“…Recently, Tyrurin and coworkers reported in a series of publications on completely abolishing acetate production in an undisclosed Clostridium strain, in favor of acetone [75], ethanol [76,77], butanol [78], mevalonate [78], methanol, or formate [79] production during autotrophic growth on syngas. However, eliminating acetate synthesis by deletion of the phosphotransacetylase and/or acetate kinase (ack) genes presumably prevents the concurrent synthesis of ATP (via SLP) by Ack.…”

Expression of amplified synthetic ethanol pathway integrated usingTn7-tool and powered at the expense of eliminatedpta,ack,spo0A and spo0J during continuous syngas or CO2/H2blend fermentation

“…; Berzin et al . ) downstream. The third component of the cassette was a 456‐bp fragment (1377318…1377774) just downstream of the pta (NC_014328, region 1376316…1377317) flanked by terminator 2 (NC_014328, region 8965…9061).…”

“…; Berzin et al . ) upstream of a synthetic Cm‐resistance gene comprising cat (region 13456…14106 of FM201786) under the promoter sequence (region 2156 to 2233 of NC_014328) flanked with lox71 (Leibig et al . ; Berzin et al .…”

“…Additional boost of the engineered cell energy pool towards ethanol path was achieved via elimination of early sporulation spo 0A and s po 0J genes using the same approach (Berzin et al . ).…”

“…As no known commercial biocatalysis utilizes naturally occurring strains, metabolic engineering renders new synthetic pathways stably functional in engineered biocatalyst micro‐organisms where the proper management of cell energy pool has been elaborated (Berzin et al . ,b).…”