Some bacterial strains such as Komagataeibacter xylinus are able to produce cellulose as an extracellular matrix. In comparison to wood-based cellulose, bacterial cellulose (BC) holds interesting properties such as biodegradability, high purity, water-holding capacity, and superior mechanical and structural properties. Aiming toward improvement in BC production titer and tailored alterations to the BC film, we engineered K. xylinus to overexpress partial and complete bacterial cellulose synthase operon that encodes activities for BC production. The changes in cell growth, end metabolite, and BC production titers from the engineered strains were compared with the wild-type K. xylinus. Although there were no significant differences between the growth of wild-type and engineered strains, the engineered K. xylinus strains demonstrated faster BC production, generating 2-4-fold higher production titer (the highest observed titer was obtained with K. xylinus-bcsABCD strain producing 4.3 ± 0.46 g/L BC in 4 days). The mechanical and structural characteristics of cellulose produced from the wild-type and engineered K. xylinus strains were analyzed with a stylus profilometer, in-house built tensile strength measurement system, a scanning electron microscope, and an X-ray diffractometer. Results from the profilometer indicated that the engineered K. xylinus strains produced thicker BC films (wild type, 5.1 μm, and engineered K. xylinus strains, 6.2-10.2 μm). Scanning electron microscope revealed no principal differences in the structure of the different type BC films. The crystallinity index of all films was high (from 88.6 to 97.5%). All BC films showed significant piezoelectric response (5.0-20 pC/N), indicating BC as a promising sensor material.

BackgroundTriacylglycerols are used in various purposes including food applications, cosmetics, oleochemicals and biofuels. Currently the main sources for triacylglycerol are vegetable oils, and microbial triacylglycerol has been suggested as an alternative for these. Due to the low production rates and yields of microbial processes, the role of metabolic engineering has become more significant. As a robust model organism for genetic and metabolic studies, and for the natural capability to produce triacylglycerol, Acinetobacter baylyi ADP1 serves as an excellent organism for modelling the effects of metabolic engineering for energy molecule biosynthesis.ResultsBeneficial gene deletions regarding triacylglycerol production were screened by computational means exploiting the metabolic model of ADP1. Four deletions, acr1, poxB, dgkA, and a triacylglycerol lipase were chosen to be studied experimentally both separately and concurrently by constructing a knock-out strain (MT) with three of the deletions. Improvements in triacylglycerol production were observed: the strain MT produced 5.6 fold more triacylglycerol (mg/g cell dry weight) compared to the wild type strain, and the proportion of triacylglycerol in total lipids was increased by 8-fold.ConclusionsIn silico predictions of beneficial gene deletions were verified experimentally. The chosen single and multiple gene deletions affected beneficially the natural triacylglycerol metabolism of A. baylyi ADP1. This study demonstrates the importance of single gene deletions in triacylglycerol metabolism, and proposes Acinetobacter sp. ADP1 as a model system for bioenergetic studies regarding metabolic engineering.

BackgroundWax esters are industrially relevant molecules exploited in several applications of oleochemistry and food industry. At the moment, the production processes mostly rely on chemical synthesis from rather expensive starting materials, and therefore solutions are sought from biotechnology. Bacterial wax esters are attractive alternatives, and especially the wax ester metabolism of Acinetobacter sp. has been extensively studied. However, the lack of suitable tools for rapid and simple monitoring of wax ester metabolism in vivo has partly restricted the screening and analyses of potential hosts and optimal conditions.ResultsBased on sensitive and specific detection of intracellular long-chain aldehydes, specific intermediates of wax ester synthesis, bacterial luciferase (LuxAB) was exploited in studying the wax ester metabolism in Acinetobacter baylyi ADP1. Luminescence was detected in the cultivation of the strain producing wax esters, and the changes in signal levels could be linked to corresponding cell growth and wax ester synthesis phases.ConclusionsThe monitoring system showed correlation between wax ester synthesis pattern and luminescent signal. The system shows potential for real-time screening purposes and studies on bacterial wax esters, revealing new aspects to dynamics and role of wax ester metabolism in bacteria.

BackgroundFatty aldehydes are industrially relevant compounds, which also represent a common metabolic intermediate in the microbial synthesis of various oleochemicals, including alkanes, fatty alcohols and wax esters. The key enzymes in biological fatty aldehyde production are the fatty acyl-CoA/ACP reductases (FARs) which reduce the activated acyl molecules to fatty aldehydes. Due to the disparity of FARs, identification and in vivo characterization of reductases with different properties are needed for the construction of tailored synthetic pathways for the production of various compounds.ResultsFatty aldehyde production in Acinetobacter baylyi ADP1 was increased by the overexpression of three different FARs: a native A. baylyi FAR Acr1, a cyanobacterial Aar, and a putative, previously uncharacterized dehydrogenase (Ramo) from Nevskia ramosa. The fatty aldehyde production was followed in real-time inside the cells with a luminescence-based tool, and the highest aldehyde production was achieved with Aar. The fate of the overproduced fatty aldehydes was studied by measuring the production of wax esters by a native downstream pathway of A. baylyi, for which fatty aldehyde is a specific intermediate. The wax ester production was improved with the overexpression of Acr1 or Ramo compared to the wild type A. baylyi by more than two-fold, whereas the expression of Aar led to only subtle wax ester production. The overexpression of FARs did not affect the length of the acyl chains of the wax esters.ConclusionsThe fatty aldehyde production, as well as the wax ester production of A. baylyi, was improved with the overexpression of a key enzyme in the pathway. The wax ester titer (0.45 g/l) achieved with the overexpression of Acr1 is the highest reported without hydrocarbon supplementation to the culture. The contrasting behavior of the different reductases highlight the significance of in vivo characterization of enzymes and emphasizes the possibilities provided by the diversity of FARs for pathway and product modulation.Electronic supplementary materialThe online version of this article (10.1186/s12934-018-0869-z) contains supplementary material, which is available to authorized users.

Bacterial cellulose (BC) is a biodegradable polymer that benefits in purity, crystallinity and superior optical, structural and mechanical properties. Such properties facilitate BC to replace the conventional non-biodegradable materials used, for instance, in sensing applications. However, BC production is largely conducted in conventional medium containing model substrates and complex carbon-containing compounds. Aiming towards the production of eco-friendly piezoelectric-responsive BC films, we isolated and characterized a novel bacterial strain affiliated to Komagataeibacter rhaeticus. The K. rhaeticus ENS9a strain synthesized BC in minimal medium containing crude glycerol, generating a titer of 2.9 ± 0.3 g/L BC. This is, to the best of our knowledge, the highest BC titer reported from an unoptimized minimal medium containing crude glycerol. Interestingly, the films prepared from crude glycerol showed normal force and bending mode sensitivities of 6–11 pC/N and 40–71 pC/N, respectively, demonstrating a green platform to address both bioprocess waste valorization and implementation of cellulose-based alternatives for the non-sustainable and non-biodegradable materials, such as fluoropolymers or lead containing piezoceramics, used in sensing applications. In silico genome analysis predicted genes partaking in carbohydrate metabolism, BC biogenesis, and nitrogen fixation/regulation. Graphic abstract