Formaldehyde degradation in Corynebacterium glutamicum involves acetaldehyde dehydrogenase and mycothiol-dependent formaldehyde dehydrogenase

Abstract

Corynebacterium glutamicum, a Gram-positive soil bacterium belonging to the actinomycetes, is able to degrade formaldehyde but the enzyme(s) involved in this detoxification process were not known. Acetaldehyde dehydrogenase Ald, which is essential for ethanol utilization, and FadH, characterized here as NAD-linked mycothiol-dependent formaldehyde dehydrogenase, were shown to be responsible for formaldehyde oxidation since a mutant lacking ald and fadH could not oxidize formaldehyde resulting in the inability to grow when formaldehyde was added to the medium. Moreover, C. glutamicum DaldDfadH did not grow with vanillate, a carbon source giving rise to intracellular formaldehyde. FadH from C. glutamicum was purified from recombinant Escherichia coli and shown to be active as a homotetramer. Mycothiol-dependent formaldehyde oxidation revealed K m values of 0.6 mM for mycothiol and 4.3 mM for formaldehyde and a V max of 7.7 U mg "1. FadH from C. glutamicum also possesses zinc-dependent, but mycothiolindependent alcohol dehydrogenase activity with a preference for short chain primary alcohols such as ethanol (K m 5330 mM, V max 59.6 U mg "1 ), 1-propanol (K m 5150 mM, V max 55 U mg "1 ) and 1-butanol (K m 550 mM, V max 50.8 U mg "1). Formaldehyde detoxification system by Ald and mycothiol-dependent FadH is essential for tolerance of C. glutamicum to external stress by free formaldehyde in its habitat and for growth with natural substrates like vanillate, which are metabolized with concomitant release of formaldehyde.

Metabolic Engineering of Corynebacterium glutamicum for Methanol Metabolism

“…ND, not determined. CO 2 (22,23). This approach should prevent loss of methanolderived formaldehyde as CO 2 and boost formaldehyde assimilation.…”

“…The dissimilatory pathway also serves as "safety valve" in case of an intracellular accumulation of formaldehyde to toxic amounts. Since the wild-type C. glutamicum already possesses an endogenous pathway for the oxidation of naturally occurring cytotoxic formaldehyde to CO 2 (22,23), we decided to initially express the genes of the synthetic methanol assimilation pathway in this strain. Two plasmids allowing for heterologous gene expression under the control of the IPTG-inducible promoter Ptac were used: one for the expression of the methanol oxidation modules [CgadhA or Bm(mdh-act)] and one for the expression of the formaldehyde assimilation modules [Bs(hpsphi) or Mg(hps-phi)].…”

“…Nonetheless, we evaluated the implementation of the NAD ϩ -dependent methanol dehydrogenase from B. methanolicus for increasing the methanol oxidation rate. Since wild-type C. glutamicum already possesses an endogenous pathway for the detoxification of naturally occurring cytotoxic formaldehyde to CO 2 (22,23), only a pathway for assimilation of formaldehyde into biomass was required. Heterologous expression of genes for HPS and PHI from methylotrophic organisms, both key enzymes of the RuMP) pathway, has already been performed to confer the capability of formaldehyde assimilation to Burkholderia cepacia and Pseudomonas putida, respectively (20,52,53).…”

“…The high availability and low market price of methanol raise the question of whether this C 1 compound could also serve as alternative carbon source for microbial production processes (20,21). Although C. glutamicum harbors an endogenous pathway for oxidation of methanol to CO 2 (22,23), it is a nonmethylotrophic organism and therefore is not able to utilize C 1 compounds as the sole carbon and energy source.…”

A new metabolic route for the production of gamma-aminobutyric acid by Corynebacterium glutamicum from glucose

“…Catabolite repression is an unusual phenomenon in C. glutamicum which is known for its ability to co-utilize a wide range of carbon sources (Blombach and Seibold 2010;Heider and Wendisch 2015). The non-growth substrate serine is co-utilized with glucose (Netzer et al 2004b), while the non-growth substrate methanol (Lessmeier et al 2013) is subject to glucose repression by RamA (Witthoff et al 2013). In addition to methanol and ethanol catabolism, also utilization of glutamate (Kramer et al 1990) is known to be repressed by glucose and is among the rare examples of catabolite repression in C. glutamicum.…”