Over Enzymen, span de natuur voor je karretje

Avondlezing georganiseerd door de Groningse Chemische Kring.

Microbial production of bulk chemicals

Samenvatting
Microorganisms perform thousands of reactions at the same time. This ability can be used for the production of chemicals.

The development of microbial conversion process is a multidisciplinary endeavor: genetic, protein, metabolic, reactor and separation engineering are all required to reach the required results. 10-15 years ago most improvements were based on reactor and separation engineering. The recent maturation molecular biology techniques and especially the development of genome editing tools as CRISPR-Cas9 resorts in a shift to metabolic engineering to tackle the challenges.

I will present 3 cases in which I analysed the process requirements for the production of a certain product, based on the chemical and physical characteristic of this chemical, and based on that, developed a microbial cell factory.

Below you can find the abstracts of the three papers I am going to discuss.

Monascus ruber as cell factory for lactic acid production at low pH
A Monascus ruber strain was isolated that was able to grow on mineral medium at high sugar concentrations and 175 g/l lactic acid at pH 2.8. Its genome and transcriptomes were sequenced and annotated. Genes encoding lactate dehydrogenase (LDH) were introduced to accomplish lactic acid production and two genes encoding pyruvate decarboxylase (PDC) were knocked out to subdue ethanol formation. The strain preferred lactic acid to glucose as carbon source, which hampered glucose consumption and therefore also lactic acid production. Lactic acid consumption was stopped by knocking out 4 cytochrome-dependent LDH (CLDH) genes, and evolutionary engineering was used to increase the glucose consumption rate. Application of this strain in a fed-batch fermentation resulted in a maximum lactic acid titer of 190 g/l at pH 3.8 and 129 g/l at pH 2.8, respectively 1.7 and 2.2 times higher than reported in literature before. Yield and productivity were on par with the best strains described in literature for lactic acid production at low pH.

Ethyl acetate production by the elusive alcohol acetyltransferase from yeast
Ethyl acetate is an industrially relevant ester that is currently produced exclusively through unsustainable processes. Many yeasts are able to produce ethyl acetate, but the main responsible enzyme has remained elusive, hampering the engineering of novel production strains. Here we describe the discovery of a new enzyme (Eat1) from the yeast Wickerhamomyces anomalus that resulted in high ethyl acetate production when expressed in Saccharomyces cerevisiae and Escherichia coli. Purified Eat1 showed alcohol acetyltransferase activity with ethanol and acetyl-CoA. Homologs of eat1 are responsible for most ethyl acetate synthesis in known ethyl acetate-producing yeasts, including S. cerevisiae, and are only distantly related to known alcohol acetyltransferases. Eat1 is therefore proposed to compose a novel alcohol acetyltransferase family within the alpha/beta hydrolase superfamily. The discovery of this novel enzyme family is a crucial step towards the development of biobased ethyl acetate production and will also help in selecting improved S. cerevisiae brewing strains.

Biocatalytic, one-pot diterminal oxidation and esterification of n-alkanes for production of a,?-diol and a,?-dicarboxylic acid esters
Direct and selective terminal oxidation of medium-chain n-alkanes is a major challenge in chemistry. Efforts to achieve this have so far resulted in low specificity and overoxidized products. Biocatalytic oxidation of medium-chain n-alkanes with for example the alkane monooxygenase AlkB from P. putida GPo1- on the other hand is highly selective. However, it also results in overoxidation. Moreover, diterminal oxidation of medium-chain n-alkanes is inefficient. Hence, α,ω-bifunctional monomers are mostly produced from olefins using energy intensive, multi-step processes.

By combining biocatalytic oxidation with esterification we drastically increased diterminal oxidation upto 92 mol% and reduced overoxidation to 3% for n-hexane. This methodology allowed us to convert medium-chain n-alkanes into α,ω-diacetoxyalkanes and esterified α,ω-dicarboxylic acids. We achieved this in a one-pot reaction with resting-cell suspensions of genetically engineered Escherichia coli.

The combination of terminal oxidation and esterification constitutes a versatile toolbox to produce α,ω-bifunctional monomers from n-alkanes.

Loopbaan dr. Ruud Alexander Weusthuis (4 dec. 1965)

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