Developing a role for Rhizopus oryzae in the biobased economy by aiming at ethanol and cyanophycin coproduction

Fossil fuels, including oil, natural gas, and coal are primary energy sources and account for 86% of the world’s energy consumption. These fossil fuels are also used as a feedstock for the production of chemicals and materials. It took millions of years to form these fuels from biomass and their consumption is therefore considered non-renewable. At a certain moment the reserves of these fossil resources will be depleted and prior to that moment mining for bulk use will be economically unfeasible. Beside the finite reserves there are also many negative effects on the environment. These include the pollution of the environment and global warming. It is generally considered that global warming is caused by the release of greenhouse gasses, such as CH4, NOx, water vapour and CO2 into the atmosphere. Alternative resources for the generation of heat and power can be formed by geothermal, water, wind, and solar energy. However, these resources do not form an alternative for the production of chemicals and materials since this requires (chemical) building blocks.Biomass can form a suitable alternative resource for the formation of these building blocks. Biomass in its pure form is complex and can in most instances not directly be used for the production of renewable compounds. Just like fossil fuels it should be processed or biorefined to obtain building blocks for chemicals and materials. In the biorefining process all the non-food fractions such as the oils, gums, carbohydrates, proteins, and lignin should be used for their most energetically favourable applications.Nitrogen containing molecules are used in many chemical applications like the production of nylon, polyurethane and acrylonitrile. Naphtha does not comprise nitrogen-containing molecules and they have to be synthesised by binding nitrogen derived from air, a very energy intensive process. Forty percent of the integral cost price of nitrogen containing chemicals from fossil resources is formed by the energy and process costs.Biomass, on the other hand, contains nitrogen containing chemicals in the form amino acids. Traditionally, commercially available amino acids are obtained through microbial fermentations. These production volumes are generally low and the price is too high for bulk use. Therefore, alternative sources need to be investigated which can supply large volumes of amino acids at low cost. Interesting sources are the residual protein streams from the food industry or the production of biofuels. These streams are however heterogeneous in composition and result in low amino acid concentrations. Cyanophycin (CPG) is naturally accumulated by cyanobacteria as insoluble granules which form an energy and nitrogen reserve material and consists of equimolar amounts of arginine and aspartic acid. The insoluble nature can be used to isolate and concentrate the granules. The wish to produce bioethanol from (lignocellulosic) waste streams coupled to the concentration and subsequent conversion of amino acids into CGP culminated into the N-ergy project. The research described in this thesis was focused on the production of cyanophycin (CGP) in the filamentous fungus Rhizopus oryzae.Chapter 2 is an in-depth analysis of the potential of R. oryzae for the production of platform chemicals and the genetic accessibility. Strains of R. oryzae are capable of producing a wide range of lignocellulosic hydrolysing enzymes and grow on a wide range of carbon sources. Using these carbon sources it is capable of producing L-(+)-lactic acid, ethanol with a fumaric acid. The yields using D-glucose are in excess of 85% of the theoretical yield for L-(+)-lactic acid and ethanol and over 65% for fumaric acid. The hydrolytic capacity and high yields of fermentation end products make this an interesting organism for the biobased economy. Though, genetic modification is challenging since the introduced DNA rarely integrates in the genome leading to mitotically unstable phenotype.In order to produce cyanophycin several cyanophycin synthetase encoding genes were introduced in the sequenced strain R. oryzae 99-880. The genes originated from Synechocystis sp. strain PCC6803 and Anabaena sp. strain PCC7120. Also, a codon optimized version of the latter gene was introduced (chapter 3). Only one transformant (expressing chpA6803) out of a total of 92 isolates was able to produce water-soluble CGP (0.5%) with traces of water-insoluble CGP. There was no correlation between transcript levels of cphA, enzyme activity, and CGP accumulation. In addition, the water soluble CGP did not contain L-lysine which is generally assumed to be the cause for the soluble behaviour. The total amount of CGP and enzyme activity is low in comparison the other genetically modified microorganisms expressing cphA’s.The common methods to determine the amino acid composition of protein samples require an acid hydrolysis followed by a derivatisation step with costly reactants. Cost effective derivatisation methods are available yet these require manual handling prior to the analysis of each individual sample. In order to analyse large volume of samples, a cost effective and fast new method was required. A new method was developed using acid hydrolysis and automated pro-column derivatisation using o-phthalaldehyde/ethanethiol reagent in combination with 9-fluorenylmethyl chloroformate (chapter 4). Due to the automated derivatisation in the injection needle the handling time is greatly reduced. Additionally, the run time for is sample using the UPLC method was 16 minutes which is a reduction of roughly 50% in comparison to a HPLC separation. The method was used for the analysis of proteins like CGP and has a mean recovery of the amino acids of 95%. With the current available techniques, it is not possible to further reduce the time required for the derivatisation and analysis.Introduction of CphA is R. oryzae was successful but resulted only in the accumulation of trace amounts of soluble cyanophycin. A possible explanation for the low accumulation is the fact that the production of soluble cyanophycin has a detrimental effect on growth of R. oryzae. We therefore tested the introduction of heterologous genes that should have a beneficial effect on growth. R. oryzae 99-880 has no xylanolytic capacity although it can grow on xylose. In order to increase the xylanolytic activity a β-1,4-endoxylanase encoding gene (xynB) of Aspergillus niger CBS 513.88 was introduced (chapter 5) using the same method as employed for the introduction of cphA’s. In total 13 transformants were isolated of which three displayed enzyme activity and one was mitotically stable. In this experiment there is a clear correlation between transcript levels of xynB and enzyme activity. The stable transformant was able to grow on xylan as carbon source. This demonstrated that metabolic engineering using the selected transformation system can be performed successfully. The large difference in numbers between transformants that successful produce β-1,4-endoxylanase in comparison to cyanophycin is indicative that the bottleneck in CGP production is product related.In recent years little development is published in respect to the genetic engineering of R. oryzae (chapter 6). The systems that are published are not always used to express genes and some are developments from the system that was also used in this study. Still R. oryzae is widely studied as an organism of interest for the biobased economy due to the hydrolytic capacity and range of product that can be formed by the various strains.