Production of 2-Hydroxyisobutyric Acid from Methanol by Methylobacterium extorquens AM1 Expressing ( R )-3-Hydroxybutyryl Coenzyme A-Isomerizing Enzymes

Abstract

The biotechnological production of the methyl methacrylate precursor 2-hydroxyisobutyric acid (2-HIBA) via bacterial poly-3-hydroxybutyrate (PHB) overflow metabolism requires suitable (R)-3-hydroxybutyryl coenzyme A (CoA)-specific coenzyme B 12 -dependent mutases (RCM). Here, we characterized a predicted mutase from Bacillus massiliosenegalensis JC6 as a mesophilic RCM closely related to the thermophilic enzyme previously identified in Kyrpidia tusciae DSM 2912 (M.-T. Weichler et al., Appl Environ Microbiol 81:4564 -4572, 2015, https://doi.org/10.1128/ AEM.00716-15). Using both RCM variants, 2-HIBA production from methanol was studied in fed-batch bioreactor experiments with recombinant Methylobacterium extorquens AM1. After complete nitrogen consumption, the concomitant formation of PHB and 2-HIBA was achieved, indicating that both sets of RCM genes were successfully expressed. However, although identical vector systems and incubation conditions were chosen, the metabolic activity of the variant bearing the RCM genes from strain DSM 2912 was severely inhibited, likely due to the negative effects caused by heterologous expression. In contrast, the biomass yield of the variant expressing the JC6 genes was close to the wild-type performance, and 2-HIBA titers of 2.1 g liter Ϫ1 could be demonstrated. In this case, up to 24% of the substrate channeled into overflow metabolism was converted to the mutase product, and maximal combined 2-HIBA plus PHB yields from methanol of 0.11 g g Ϫ1 were achieved. Reverse transcription-quantitative PCR analysis revealed that metabolic genes, such as methanol dehydrogenase and acetoacetyl-CoA reductase genes, are strongly downregulated after exponential growth, which currently prevents a prolonged overflow phase, thus preventing higher product yields with strain AM1.IMPORTANCE In this study, we genetically modified a methylotrophic bacterium in order to channel intermediates of its overflow metabolism to the C 4 carboxylic acid 2-hydroxyisobutyric acid, a precursor of acrylic glass. This has implications for biotechnology, as it shows that reduced C 1 substrates, such as methanol and formic acid, can be alternative feedstocks for producing today's commodities. We found that product titers and yields depend more on host physiology than on the activity of the introduced heterologous function modifying the overflow metabolism. In addition, we show that the fitness of recombinant strains substantially varies when they express orthologous genes from different origins. Further studies are needed to extend the overflow production phase in methylotrophic microorganisms for the implementation of biotechnological processes.KEYWORDS acyl-CoA mutase, bulk chemicals, fed-batch bioreactor, overflow metabolism, polyhydroxybutyrate

Transposon Sequencing Uncovers an Essential Regulatory Function of Phosphoribulokinase for Methylotrophy

“…Taken together, the generated essentiality data extend our understanding of methylotrophy. At the same time, the data provide a basis for metabolic engineering to convert methanol into value-added products in M. extorquens itself [50,[52][53][54] and beyond for transplanting methylotrophy to established platform organisms [6,7].…”

Production of 3-hydroxypropionic acid in engineered Methylobacterium extorquens AM1 and its reassimilation through a reductive route

“…Methylobacterium extorquens AM1 has been considered as a potential platform strain for industrial production of valuable chemicals such as mevalonate, 1-butanol and 2-hydroxyisobutyrate [ 30 , 32 , 33 , 41 ]. In this work, we first optimized a 3-HP synthetic pathway in M. extorquens AM1, and then focused on the demonstration of the mechanism of 3-HP reassimilation.…”

A Review of the Biotechnological Production of Methacrylic Acid

“…Biocatalysis reliant on B12 dependent enzymes often require vitamin supplementation, and can suffer from enzyme inactivation, requiring reactivation (Daniel et al, 1998;Mori and Toraya, 1999). To date 2-HIBA biosynthesis has been reported in engineered microbes at titers as high as 6.4g/L (Burgard et al, 2009;Hoefel et al, 2010;Reinecke et al, 2011;Soucaille and Boisart, 2011;Rohde et al, 2017).…”