Engineering Corynebacterium glutamicum to produce the biogasoline isopentenol from plant biomass hydrolysates

Abstract

Background: Many 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. Results: We 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. Conclusions: We describe the successful expression of a heterologous mevalonate-based pathway in the Grampositive 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.

Genome-scale metabolic rewiring improves titers rates and yields of the non-native product indigoidine at scale

“…Since the MCS-based approach requires the delineation of specific growth conditions, such as starting carbon source, we examined if the gene cut set with glucose as a substrate could maintain product pairing with other known native carbon substrates for P. putida , such as para -coumarate and lysine 17 , 22 . These substrates are important carbon streams that could be utilized from lignocellulosic biomass hydrolysates 23 , 24 . FBA with these alternate carbon sources (i.e., lysine, para- coumarate) indicated that a strain engineered using the 16-gene cMCS strategy for the glucose would fail to produce glutamine (Supplementary Table 4 ).…”

Growth-uncoupled isoprenoid synthesis in Rhodobacter sphaeroides

“…The relatively small increase in flux through the MVA pathway indicated that this pathway benefited from the increased amounts of AA-CoA and NADPH, but its capacity is limiting isoprenoid production. One of the rate-determining enzymes of the heterologous pathway is HMG-CoA reductase, already described as crucial enzyme for enhancing isoprenoid flux [15,39]. We speculate that improving the catalytic efficiency of this enzyme might allow the MVA pathway to benefit from the higher availability of NADPH and AA-CoA.…”

Growth-uncoupled isoprenoid synthesis in Rhodobacter sphaeroides

“…The relatively small increase in ux through the MVA pathway indicated that this pathway bene ted from the increased amounts of AA-CoA and NADPH, but its capacity is limiting isoprenoid production. One of the rate-determining enzymes of the heterologous pathway is HMG-CoA reductase, already described as crucial enzyme for enhancing isoprenoid ux (15,39). We speculate that improving the catalytic e ciency of this enzyme might allow the MVA pathway to bene t from the higher availability of NADPH and AA-CoA.…”