High-level recombinant protein production with Corynebacterium glutamicum using acetate as carbon source.

In recent years, biotechnological conversion of the alternative carbon source acetate has attracted much attention. So far, acetate has been mainly used for microbial production of bioproducts with bulk applications. In this study, we aimed to investigate the potential of acetate as carbon source for heterologous protein production using the acetate-utilizing platform organism Corynebacterium glutamicum. For this purpose, expression of model protein eYFP with the promoter systems T7lac and tac was characterized during growth of C. glutamicum on acetate as sole carbon source. The results indicated a 3.3-fold higher fluorescence level for acetate-based eYFP production with T7 expression strain MB001(DE3) pMKEx2-eyfp compared to MB001 pEKEx2-eyfp. Interestingly, comparative eyfp expression studies on acetate or glucose revealed an up to 83% higher biomass-specific production for T7 RNAP-dependent eYFP production using acetate as carbon source. Furthermore, high-level protein accumulation on acetate was demonstrated for the first time in a high cell density cultivation process with pH-coupled online feeding control, resulting in a final protein titer of 2.7 g/L and product yield of 4 g per 100 g cell dry weight. This study presents a first proof of concept for efficient microbial upgrading of potentially low-cost acetate into high-value bioproducts, such as recombinant proteins.

Metabolic engineering of Corynebacterium glutamicum for acetate-based itaconic acid production.

BACKGROUND: Itaconic acid is a promising platform chemical for a bio-based polymer industry. Today, itaconic acid is biotechnologically produced with Aspergillus terreus at industrial scale from sugars. The production of fuels but also of chemicals from food substrates is a dilemma since future processes should rely on carbon sources which do not compete for food or feed. Therefore, the production of chemicals from alternative substrates such as acetate is desirable to develop novel value chains in the bioeconomy. RESULTS: In this study, Corynebacterium glutamicum ATCC 13032 was engineered to efficiently produce itaconic acid from the non-food substrate acetate. Therefore, we rewired the central carbon and nitrogen metabolism by inactivating the transcriptional regulator RamB, reducing the activity of isocitrate dehydrogenase, deletion of the gdh gene encoding glutamate dehydrogenase and overexpression of cis-aconitate decarboxylase (CAD) from A. terreus optimized for expression in C. glutamicum. The final strain C. glutamicum ΔramB Δgdh IDHR453C (pEKEx2-malEcadopt) produced 3.43 ± 0.59 g itaconic acid L-1 with a product yield of 81 ± 9 mmol mol-1 during small-scale cultivations in nitrogen-limited minimal medium containing acetate as sole carbon and energy source. Lowering the cultivation temperature from 30 °C to 25 °C improved CAD activity and further increased the titer and product yield to 5.01 ± 0.67 g L-1 and 116 ± 15 mmol mol-1, respectively. The latter corresponds to 35% of the theoretical maximum and so far represents the highest product yield for acetate-based itaconic acid production. Further, the optimized strain C. glutamicum ΔramB Δgdh IDHR453C (pEKEx2-malEcadopt), produced 3.38 ± 0.28 g itaconic acid L-1 at 25 °C from an acetate-containing aqueous side-stream of fast pyrolysis. CONCLUSION: As shown in this study, acetate represents a suitable non-food carbon source for itaconic acid production with C. glutamicum. Tailoring the central carbon and nitrogen metabolism enabled the efficient production of itaconic acid from acetate and therefore this study offers useful design principles to genetically engineer C. glutamicum for other products from acetate.

Acetate-based production of itaconic acid with Corynebacterium glutamicum using an integrated pH-coupled feeding control.

To date, most bio-based products of industrial biotechnology stem from sugar-based carbon sources originating from food and feed competing resources. Exemplary for bioproducts converted from glucose, the potential C5 platform chemical itaconic acid is presently produced by the filamentous fungus Aspergillus terreus. Here, an engineered strain of the industrial platform organism Corynebacterium glutamicum ATCC 13032 was used for acetate-based production of itaconic acid to overcome current production difficulties. For this purpose, C. glutamicum ICDR453C (pEKEx2-malEcadopt) with a mutated icd variant for reduced isocitrate dehydrogenase activity was constructed harbouring pEKEx2-malEcadopt, that includes a cis-aconitate dehydrogenase gene originating from A. terreus. Overall, a peak volumetric productivity of 1.01 gL-1h-1 was achieved resulting in an itaconate titer of 29.2 g/L, by using an integrated pH-coupled acetate feeding control in a fed-batch process without base titration. The results support the high potential of acetate as alternative substrate for bioproduction.

High cell density cultivation of Corynebacterium glutamicum on bio-based lignocellulosic acetate using pH-coupled online feeding control.

Acetate represents a promising alternative carbon source for future industrial biotechnology. In this study, the high potential of Corynebacterium glutamicum for utilizing acetate as sole carbon source was demonstrated. Batch culture studies revealed that C. glutamicum ATCC 13032 naturally exhibits high acetate tolerance with maximum growth rates (µmax = 0.47 h-1) similar to those on D-glucose. Based on a simple and auto-regulated pH-coupled feeding strategy which utilizes bio-acetic acid in pure form, a novel and high-efficient fed-batch process was developed in a 42 L stirred-tank bioreactor. By optimizing the carbon-to-nitrogen (C/N) feeding ratio, maximum biomass concentrations of 80.2 gCDW/L were achieved with a space-time yield of 66.6 gCDW/L·d. In addition, a process model was implemented describing the time-courses of biomass growth and substrate concentrations. This is the first study in which an industrial platform organism was grown to high cell densities using green, lignocellulosic acetate as an alternative carbon source.

From Acetate to Bio-Based Products: Underexploited Potential for Industrial Biotechnology.

Currently, most biotechnological products are based on microbial conversion of carbohydrate substrates that are predominantly generated from sugar- or starch-containing plants. However, direct competitive uses of these feedstocks in the food and feed industry represent a dilemma, so using alternative carbon sources has become increasingly important in industrial biotechnology. A promising alternative carbon source that may be generated in substantial amounts from lignocellulosic biomass and C1 gases is acetate. This review discusses the underexploited potential of acetate to become a next-generation platform substrate in future industrial biotechnology and summarizes alternative sources and routes for acetate production. Furthermore, biotechnological aspects of microbial acetate utilization and the state of the art of biotechnological acetate conversion into value-added bioproducts are highlighted.

Time-course and degradation rate of membrane scaffold protein (MSP1D1) during recombinant production.

Membrane scaffold proteins (MSPs) are synthetic derivatives of apolipoprotein A-I, a major protein component of human high-density lipoprotein complexes. The most common among these is the variant MSP1D1, which has been in the focus of research on membrane mimetics in the past. As such, the amphipathic MSP1D1 has the ability to self-assemble in the presence of synthetic phospholipids into discoidal nanoparticles, so called nanodiscs. The recombinant production of MSP is exclusively reported using a standard laboratory expression system of the pET family. However, strong variations in both yield and achieved concentration as well as complications related to unspecific degradation are commonly reported. In addition, the time-course of recombinant protein as well as specific protein yields have not yet been quantified conclusively. In this study, the time-course of MSP1D1 concentration was investigated in a standard pET expression system in terms of quantification of production and degradation rates in comparison to a reference protein (eGFP).