Abstract
BackgroundMany microbes used for the rapid discovery and development of metabolic pathways have sensitivities to final products and process reagents. Isopentenol (3-methyl-3-buten-1-ol), a biogasoline candidate, has an established heterologous gene pathway but is toxic to several microbial hosts. Reagents used in the pretreatment of plant biomass, such as ionic liquids, also inhibit growth of many host strains. We explored the use of Corynebacterium glutamicum as an alternative host to address these constraints.ResultsWe found C. glutamicum ATCC 13032 to be tolerant to both the final product, isopentenol, as well to three classes of ionic liquids. A heterologous mevalonate-based isopentenol pathway was engineered in C. glutamicum. Targeted proteomics for the heterologous pathway proteins indicated that the 3-hydroxy-3-methylglutaryl-coenzyme A reductase protein, HmgR, is a potential rate-limiting enzyme in this synthetic pathway. Isopentenol titers were improved from undetectable to 1.25 g/L by combining three approaches: media optimization; substitution of an NADH-dependent HmgR homolog from Silicibacter pomeroyi; and development of a C. glutamicum ∆poxB ∆ldhA host chassis.ConclusionsWe describe the successful expression of a heterologous mevalonate-based pathway in the Gram-positive industrial microorganism, C. glutamicum, for the production of the biogasoline candidate, isopentenol. We identified critical genetic factors to harness the isopentenol pathway in C. glutamicum. Further media and cultivation optimization enabled isopentenol production from sorghum biomass hydrolysates.
Highlights
Many microbes used for the rapid discovery and development of metabolic pathways have sensitivities to final products and process reagents
C. glutamicum is tolerant to three classes of ionic liquid (IL) and exogenous isopentenol We examined three broad IL classes: 1-ethyl-3-methyl imidazolium ([C2C1im]+) derived; cholinium ([Ch]+) derived; and protic: ethanolamine acetate [ETA][OAc] and diethanolamine acetate [DEOA][OAc]) in different cation/anion configurations, for toxicity against C. glutamicum
Ethanolamine- and diethanolamine-based ILs did not show a dosage dependent inhibition on C. glutamicum growth (Fig. 2c), suggesting that at tested concentrations these two representative protic ILs have no deleterious impact on C. glutamicum growth
Summary
Many microbes used for the rapid discovery and development of metabolic pathways have sensitivities to final products and process reagents. Microbial hosts used for the rapid discovery and development of metabolic pathways can have drawbacks that limit their biotechnological applications beyond the laboratory scale These disadvantages include inhibition from components of the growth media and toxicity from metabolic intermediates or final products [1]. Of particular relevance for renewable biofuel production is its capacity of simultaneously utilizing glucose and xylose, two major components of plant biomass hydrolysates [6, 7], as well as p-coumaric and ferulic acids as carbon sources [8, 9] These factors have contributed to the development of C. glutamicum as a production host for many bioproducts [10,11,12]. Isopentenol (3-methyl-3-buten-1-ol) is a prominent example of a terpene compound that is desirable as both biogasoline as well as a platform chemical and has been developed in other microbial systems (e.g., E. coli) [15], but not in C. glutamicum
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