Metabolic engineering toward 1-butanol derivatives in solvent producing clostridia

M.A.J. Siemerink

Research output: Thesisinternal PhD, WU


Chapter 1 of this thesis gives an overview about the history of the acetone, butanol and ethanol (ABE) fermentation. The responsible solventogenic clostridia with their central metabolism are briefly discussed. Despite the fact that scientific research on the key organisms of the ABE process has continued over the past decades and even increased in recent years, still numerous aspects remain unclear.
Economically, the biggest challenge within the ABE fermentation field, remains the 1-butanol toxicity. Due to its toxicity the yield of 1-butanol does not exceed 1-2% (w/v), and only little progress has been made over the past years. Nevertheless, the ABE fermentation process became interesting again, because of the global interest in biofuels or biofuel additives.
Nowadays, in an attempt to reach higher yields, other microorganisms are also being explored as hosts for the production of solvents. E. coli is often chosen as a promising host organism for the microbial production of biofuels. Nevertheless, clostridial hosts remain interesting, due to several reasons, like i) availability of a genetic system, ii) natural production of solvents and iii) relatively high tolerance towards solvents. A disadvantage of the usage of these solventogenic organisms is the inability to use cellulose and hemi-cellulose as substrate.
With respect to the production of potentially interesting 1-butanol derivatives, we focussed on 2,3-butanediol. This industrially valuable compound is already produced in nature by several bacteria, but not by C. acetobutylicum. In this thesis, the production of 2,3-butanediol by Clostridium acetobutylicum is investigated. In Chapter 1 the 2,3-butanediol biosynthesis pathway is extensively described.

The two industrially most important and sequenced solventogenic clostridia are C. acetobutylicum ATCC 824 and C. beijerinckii NCIMB 8052. A lot of biochemical information is known about the various metabolic steps of the central catabolic pathway of C. acetobutylicum ATCC 824. In Chapter 2, comparisons are made between the pathways of both species. With the genome sequence of C. beijerinckii NCIMB 8052 also available, likely candidates for the 34 involved enzymatic conversions within the central catabolic pathway of C. beijerinckii, could be predicted. The enzymatic conversions involved in glucose uptake, glycolysis, gluconeogenesis, pyruvate conversion and acetyl-CoA conversion towards the different end products are being discussed.

Chapter 3 describes and investigates a novel approach in solving the problem of the 1-butanol toxicity towards C. acetobutylicum. Increasing of the tolerance of 1-butanol has been tried more often in other research groups. In our approach, we chose for the introduction of new biosynthesis pathways that enable the production of less toxic 1-butanol derivatives. The toxicity of compounds is expected to correlate with its lipophilicity, e.g. the tendency to accumulate in cell membranes. It can be expressed as the logarithm of the partition coefficient with octanol and water (log Kow value). Chapter 3 describes the growth experiments that were performed to access the toxicity of the various derivatives that were selected, viz. iso-butanol, 2-butanol, tert-butanol, 2,3-butanediol, iso-amyl alcohol, butyl acetate, butyl butyrate and butyl lactate. Iso-butanol, 2-butanol and 2,3-butanediol emerged as likely alternatives to 1-butanol, based on their log Kow value and their behaviour in the toxicity test.

2,3-Butanediol appeared to be a potential candidate, which is less toxic to C. acetobutylicum than 1-butanol. The precursor of 2,3-butanediol, acetoin, which is already produced by C. acetobutylicum in small amounts, is formed by the decarboxylation of acetolactate. The conversion is catalyzed by acetolactate decarboxylase. In Chapter 4 the identification, heterologous production, purification and biochemical characterization of a acetolactate decarboxylase from C. acetobutylicum ATCC 824 is described. Ca-ALD encoded by CAC2967 was proven to exhibit acetolactate decarboxylase activity. Size exclusion chromatography revealed that the native enzyme mainly exists as dimer of 27 kDa subunits. Optimal activity was found around 40 °C, and at pH 5.2. The enzyme is dependent on the presence of bivalent metal ions, like Zn2+ or Co2+. The half life is estimated as 25 hours at 37 °C. The Ca-ALD binds acetolactate cooperatively with a Hill coefficient of 1.49. Also, a K1/2 of 16.8 mM and a Vmax of 51.9 U/mg was determined. Furthermore, a shuttle vector was constructed to express Ca-ald under control of the strong adc promoter in C. acetobutylicum ATCC 824. However, despite successful transformation, no significant increase in acetoin production was observed in the Ca-ALD overexpressing strain.

Only one additional enzyme is needed to complete the 2,3-butanediol biosynthesis pathway in C. acetobutylicum. This enzyme, catalyzing the reduction of acetoin to 2,3-butandiol is called an acetoin reductase. Chapter 5 describes the identification, heterologous production, purification and biochemical characterization of an acetoin reductase from C. beijerinckii. A bioinformatic screening within the genome of C. beijerinckii , revealed eight putative acetoin reductases. Out of six successfully cloned genes, one (CBEI_1464) showed substantial acetoin reductase activity after heterologous expression in E. coli. This purified enzyme (Cb-ACR) was found to exist predominantly as homodimer of 37 kDa subunits. The enzyme has a preference for NADPH (Km = 0.32 μM) as electron donor, with a specific activity amounting to 76 U. mg-1. Optimal activity was found around 68 °C, for both reactions and at pH 6.5 and 9.5, for the reduction and oxidation reaction, respectively. ICP-AES analysis revealed the presence of ~2 Zn2+ atoms and ~1 Ca2+ atom per monomer. To gain insight into the reaction mechanism, but also into the substrate- and cofactor-specificity, a structural model was constructed with a ketose reductase (sorbitol dehydrogenase) from Bemisia argentifolii (silverleaf whitefly) as template. The catalytic zinc atom is likely coordinated by Cys37, His70, Glu71 in Cb-ACR, while the structural zinc site is probably composed of Cys100, Cys103, Cys106, and Cys114.

The acetoin reductase (Cb-ACR) found in C. beijerinckii is used for in vivo experiments in C. acetobutylicum. In Chapter 6 the production of D-2,3-butanediol production in C. acetobutylicum ATCC 824 by heterologous expression of Cb-ACR is described. Under certain conditions Clostridium acetobutylicum ATCC 824 (and derived strains) generates both D and L stereoisomers of acetoin, but due to the lack of an ACR enzyme, does not produce 2,3 butanediol. A gene encoding ACR from Clostridium beijerinckii NCIMB 8052 has been functionally expressed in C. acetobutylicum under control of two strong promoters, i.e. the constitutive thl promoter and the late exponential adc promoter. Both ACR-overproducing strains have been grown in batch cultures, during which 89 90% of the natively produced acetoin has been converted to 20 22 mM D 2,3 butanediol. Addition of a racemic mixture of acetoin did lead to the production of both, D 2,3 butanediol and meso 2,3 butanediol. A metabolic network is proposed that is in agreement with the experimental data. Native 2,3 butanediol production is a first step towards a potential homo fermentative 2 butanol producing strain of C. acetobutylicum as will be discussed in this thesis.

Original languageEnglish
QualificationDoctor of Philosophy
Awarding Institution
  • Wageningen University
  • van der Oost, John, Promotor
  • Kengen, Servé, Co-promotor
Award date12 Nov 2010
Place of Publication[S.l.
Print ISBNs9789085857976
Publication statusPublished - 2010


  • industrial microbiology
  • clostridium
  • butanol
  • derivatives
  • metabolism


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