Identification of vitamin B12 producing bacteria based on the presence of bluB/cobT2 homologues.

OBJECTIVES: The objective of the study was to develop a strategy for the identification of new vitamin B12-producing species and to characterize their production capability using a fast and sensitive LC-MS/MS method developed in this study. RESULTS: Searching for homologues of the bluB/cobT2 fusion gene known to be responsible for the production of the active vitamin B12 form in P. freudenreichii was shown to be a successful strategy for the identification of new vitamin B12-producing strains. The analysis of the identified strains via LC-MS/MS showed the ability of Terrabacter sp. DSM102553, Yimella lutea DSM19828 and Calidifontibacter indicus DSM22967 to produce the active form of vitamin B12. Further analysis of vitamin B12 production capability of Terrabacter sp. DSM102553 in M9 minimal medium and peptone-based media revealed that the highest yield of 2.65 µg of vitamin B12 per g dry cell weight was obtained in M9 medium. CONCLUSIONS: The proposed strategy enabled identification of Terrabacter sp. DSM102553, whose relatively high yields obtained in the minimal medium open new perspectives for the possible application of the strain for biotechnological vitamin B12 production.

First time ß-farnesene production by the versatile bacterium Cupriavidus necator.

BACKGROUND: Terpenes are remarkably diverse natural structures, which can be formed via two different pathways leading to two common intermediates. Among those, sesquiterpenes represent a variety of industrially relevant products. One important industrially produced product is ß-farnesene as a precursor for a jet fuel additive. So far, microbial terpene production has been mostly limited to known production hosts, which are only able to grow on heterotrophic substrates. RESULTS: In this paper, we for the first time describe ß-farnesene production by the versatile bacterial host Cupriavidus necator on fructose, which is known to grow hetero- and autotrophically and even in bioelectrochemical systems. We were able to show a growth-dependent production of ß-farnesene by expressing the ß-farnesene synthase from Artemisia annua in C. necator H16 PHB-4. Additionally, we performed a scale-up in a parallel reactor system with production titers of 26.3 ± 1.3 µM ß-farnesene with a fed-batch process. CONCLUSIONS: The ß-farnesene production titers reported in this paper are not in the same range as titers published with known heterotrophic producers E. coli or S. cerevisiae. However, this proof-of-principle study with C. necator as production host opens new synthesis routes toward a sustainable economy and leaves room for further optimizations, which have been already performed with the known production strains.

Gram-scale production of the sesquiterpene α-humulene with Cupriavidus necator.

Terpenoids have an impressive structural diversity and provide valuable substances for a variety of industrial applications. Among terpenes, the sesquiterpenes (C15 ) are the largest subclass with bioactivities ranging from aroma to health promotion. In this article, we show a gram-scale production of the sesquiterpene α-humulene in final aqueous concentrations of 2 g L-1 with the recombinant strain Cupriavidus necator pKR-hum in a fed-batch mode on fructose as carbon source and n-dodecane as an extracting organic phase for in situ product removal. Since C. necator is capable of both heterotrophic and autotrophic growth, we additionally modeled the theoretically possible yields of a heterotrophic versus an autotrophic process on CO2 in industrially relevant quantities. We compared the cost-effectiveness of both processes based on a production of 10 t α-humulene per year, with both processes performing equally with similar costs and gains. Furthermore, the expression and activity of 3-hydroxymethylglutaryl-CoA reductase (hmgR) from Myxococcus xanthus was identified as the main limitation of our constructed C. necator pKR-hum strain. Thus, we outlined possible solutions for further improvement of our production strain, for example, the replacement of the hmgR from M. xanthus by a plant-based variant to increase α-humulene production titers in the future.

Cell Factory Design and Optimization for the Stereoselective Synthesis of Polyhydroxylated Compounds.

A synthetic cascade for the transformation of primary alcohols into polyhydroxylated compounds in Escherichia coli, through the in situ preparation of cytotoxic aldehyde intermediates and subsequent aldolase-mediated C-C bond formation, has been investigated. An enzymatic toolbox consisting of alcohol dehydrogenase AlkJ from Pseudomonas putida and the dihydroxyacetone-/hydroxyacetone-accepting aldolase variant Fsa1-A129S was applied. Pathway optimization was performed at the genetic and process levels. Three different arrangements of the alkJ and fsa1-A129S genes in operon, monocistronic, and pseudo-operon configuration were tested. The last of these proved to be most beneficial with regard to bacterial growth and protein expression levels. The optimized whole-cell catalyst, combined with a refined solid-phase extraction downstream purification protocol, provides diastereomerically pure carbohydrate derivatives that can be isolated in up to 91 % yield over two reaction steps.

Escherichia coli Fails to Efficiently Maintain the Activity of an Important Flavin Monooxygenase in Recombinant Overexpression.

This paper describes the measurement and analysis of in vivo activity and stability of cyclohexanone monooxygenase from Acinetobacter sp. NCIMB 9871 (CHMO), a model Baeyer-Villiger monooxygenase, in the recombinant host Escherichia coli. This enzyme was often described as poorly stable in vitro, and has recently been found to deactivate rapidly in the absence of its essential cofactors and antioxidants. Its stability in vivo was scarcely studied, so far. Under conditions common for the overexpression of CHMO we investigated the ability of the host to support these properties using metabolomics. Our results showed that E. coli failed to provide the intracellular levels of cofactors required to functionally stabilize the enzyme, although the biocatalyst was produced in high concentration, and was invariably detected after protein synthesis had stopped. We thus infer that biotechnological applications of CHMO with this host relied on a residual activity of approximately 5-10%. Other microorganisms might offer a more efficient solution for recombinant production of CHMO and related enzymes.

Non-hazardous biocatalytic oxidation in Nylon-9 monomer synthesis on a 40 g scale with efficient downstream processing.

This paper describes the development of a biocatalytic process on the multi-dozen gram scale for the synthesis of a precursor to Nylon-9, a specialty polyamide. Such materials are growing in demand, but their corresponding monomers are often difficult to synthesize, giving rise to biocatalytic approaches. Here, we implemented cyclopentadecanone monooxygenase as an Escherichia coli whole-cell biocatalyst in a defined medium, together with a substrate feeding-product removal concept, and an optimized downstream processing (DSP). A previously described hazardous peracid-mediated oxidation was thus replaced with a safe and scalable protocol, using aerial oxygen as oxidant, and water as reaction solvent. The engineered process converted 42 g (0.28 mol) starting material ketone to the corresponding lactone with an isolated yield of 70% (33 g), after highly efficient DSP with 95% recovery of the converted material, translating to a volumetric yield of 8 g pure product per liter. Biotechnol. Bioeng. 2017;114: 1670-1678. © 2017 Wiley Periodicals, Inc.

Biomass pretreatment affects Ustilago maydis in producing itaconic acid.

BACKGROUND: In the last years, the biotechnological production of platform chemicals for fuel components has become a major focus of interest. Although ligno-cellulosic material is considered as suitable feedstock, the almost inevitable pretreatment of this recalcitrant material may interfere with the subsequent fermentation steps. In this study, the fungus Ustilago maydis was used to produce itaconic acid as platform chemical for the synthesis of potential biofuels such as 3-methyltetrahydrofuran. No studies, however, have investigated how pretreatment of ligno-cellulosic biomass precisely influences the subsequent fermentation by U. maydis. Thus, this current study aims to first characterize U. maydis in shake flasks and then to evaluate the influence of three exemplary pretreatment methods on the cultivation and itaconic acid production of this fungus. Cellulose enzymatically hydrolysed in seawater and salt-assisted organic-acid catalysed cellulose were investigated as substrates. Lastly, hydrolysed hemicellulose from fractionated beech wood was applied as substrate. RESULTS: U. maydis was characterized on shake flask level regarding its itaconic acid production on glucose. Nitrogen limitation was shown to be a crucial condition for the production of itaconic acid. For itaconic acid concentrations above 25 g/L, a significant product inhibition was observed. Performing experiments that simulated influences of possible pretreatment methods, U. maydis was only slightly affected by high osmolarities up to 3.5 osmol/L as well as of 0.1 M oxalic acid. The production of itaconic acid was achieved on pretreated cellulose in seawater and on the hydrolysed hemicellulosic fraction of pretreated beech wood. CONCLUSION: The fungus U. maydis is a promising producer of itaconic acid, since it grows as single cells (yeast-like) in submerged cultivations and it is extremely robust in high osmotic media and real seawater. Moreover, U. maydis can grow on the hemicellulosic fraction of pretreated beech wood. Thereby, this fungus combines important advantages of yeasts and filamentous fungi. Nevertheless, the biomass pretreatment does indeed affect the subsequent itaconic acid production. Although U. maydis is insusceptible to most possible impurities from pretreatment, high amounts of salts or residues of organic acids can slow microbial growth and decrease the production. Consequently, the pretreatment step needs to fit the prerequisites defined by the actual microorganisms applied for fermentation.