Lactose-inducible system for metabolic engineering of Clostridium ljungdahlii

Abstract

The development of tools for genetic manipulation of Clostridium ljungdahlii has increased its attractiveness as a chassis for autotrophic production of organic commodities and biofuels from syngas and microbial electrosynthesis and established it as a model organism for the study of the basic physiology of acetogenesis. In an attempt to expand the genetic toolbox for C. ljungdahlii, the possibility of adapting a lactose-inducible system for gene expression, previously reported for Clostridium perfringens, was investigated. The plasmid pAH2, originally developed for C. perfringens with a gusA reporter gene, functioned as an effective lactose-inducible system in C. ljungdahlii. Lactose induction of C. ljungdahlii containing pB1, in which the gene for the aldehyde/alcohol dehydrogenase AdhE1 was downstream of the lactose-inducible promoter, increased expression of adhE1 30-fold over the wild-type level, increasing ethanol production 1.5-fold, with a corresponding decrease in acetate production. Lactose-inducible expression of adhE1 in a strain in which adhE1 and the adhE1 homolog adhE2 had been deleted from the chromosome restored ethanol production to levels comparable to those in the wild-type strain. Inducing expression of adhE2 similarly failed to restore ethanol production, suggesting that adhE1 is the homolog responsible for ethanol production. Lactose-inducible expression of the four heterologous genes necessary to convert acetyl coenzyme A (acetyl-CoA) to acetone diverted ca. 60% of carbon flow to acetone production during growth on fructose, and 25% of carbon flow went to acetone when carbon monoxide was the electron donor. These studies demonstrate that the lactose-inducible system described here will be useful for redirecting carbon and electron flow for the biosynthesis of products more valuable than acetate. Furthermore, this tool should aid in optimizing microbial electrosynthesis and for basic studies on the physiology of acetogenesis.

FIG 1

Pathways for indigenous formation of acetate and ethanol from acetyl-CoA and the synthetic pathway introduced for acetone (bold and italic) in C. ljungdahlii. For each mole of acetone formed, one acetyl-CoA is converted to acetate, yielding one ATP via substrate-level phosphorylation. The acetate does not accumulate but rather is recycled to form acetyl-CoA with CoA obtained from the conversion of acetoacetyl-CoA to acetoacetate, which is then decarboxylated to acetone. Abbreviations: Pta, phosphotransacetylase; Ack, acetate kinase; AdhE1, alcohol/aldehyde dehydrogenase; Thl, thiolase; CoAt, coenzyme A transferase; Aadc, acetoacetate decarboxylase.

FIG 2

Induction of β-glucuronidase activity with 1, 10, or 20 mM lactose in the C. ljungdahlii strain containing plasmid pAH2 (A) or the control pAH2-MCS (B). Cells were grown in DSMZ879 medium supplemented with 5 g/liter fructose. Data are means for duplicates and are representative of two independent experiments. x axis, time (in hours).

FIG 3

Lactose-induced expression of adhE1. Transcript abundance determined by qRT-PCR was normalized to expression of the housekeeping gene recA. Total RNA was isolated from fructose-grown mid-log-phase cells. Data are the means and standard errors from triplicate assays on two biological replicates.

FIG 4

Cell growth and production of ethanol and acetate by the C. ljungdahlii wild-type strain containing the plasmid pB1 under fructose fermentation growth conditions in DSMZ879. (A) Cell growth. (B) Ethanol production. (C) Acetate production. Data are the means and standard deviations for triplicate cultures. Where no error bar is seen, the bar is smaller than the size of the symbol.

FIG 5

Cell growth and production of ethanol and acetate by the C. ljungdahlii adhE1/adhE2-deficient mutant containing either plasmid pKRAH1 or pB1 under fructose fermentation growth condition in DSMZ879. (A and D) Cell growth; (B and E) ethanol production; (C and F) acetate production. (A, B, and C) Noninduced samples; (D, E, and F) samples induced with 1 mM lactose. Data are the means and standard deviations for triplicate cultures. Where no error bar is seen, the bar is smaller than the size of the symbol.

FIG 6

Cell growth and production of acetone and acetate by the C. ljungdahlii wild-type strain containing either plasmid pKRAH1 or pB3 under fructose fermentation growth condition in PETC1754. (A and D) Cell growth; (B and E) acetone production; (C and F) acetate production. (A, B, and C) noninduced samples; (D, E, F) samples induced with 1 mM lactose. Data are the means and standard deviations for triplicate cultures. Where no error bar is seen, the bar is smaller than the size of the symbol.

FIG 7

Cell growth and production of acetone, acetate, and ethanol by the C. ljungdahlii wild-type strain containing plasmid pB3 under autotrophic growth conditions with CO as the electron donor. (A) Cell growth; (B) acetone production; (C) acetate production; (D) ethanol production. Data are the means and standard deviations for triplicate cultures. Where no error bar is seen, the bar is smaller than the size of the symbol.