A remix in microbiology: enabling microbial ester production through the engineering of enzymes, pathways and genomes

Microbiology is an extraordinary field which has been, and will continue to be, central to the advancement of human cultures. In fact, human cultures and even our planet would not be as they are today if microorganisms and microbiology would not exist. Basic, but at the same time, complex microbiological processes play key roles in biodegradation and nutrient (re)cycling, climate change, disease and biotechnology. Due to its importance, a brief history about microbiology is provided in Chapter 1 and the related (old or new) biotechnological applications are described. The importance of the microorganism-workhorses is highlighted and a “start-at-the-end” approach is recommended for choosing the appropriate microbe for the production of the final product. A brief introduction to clostridia species and their potential for the sustainable production of ester compounds is provided. Lastly, the importance of the revolutionary CRISPR-Cas genome engineering tools is highlighted.Chapter 2 reviews the state-of-the-art technologies used for the microbial production of short and medium chain esters. Several enzymatic processes are described with main focus on the AAT enzymes. In addition, several metabolic engineering processes are reviewed, along with the physical properties of the final ester product. Lastly, an outlook is provided on other products (e.g. alcohols, carboxylic acids and diols) that can be derived from (microbially produced) esters.In Chapter 3, the unique Eat1 enzyme was further characterized using in vitro and in vivo approaches. Two new activities (alcoholysis and thiolysis) were added to the existing activities (AAT, esterase and thioesterase) of Eat1. Alcoholysis appears to be the main activity of the enzyme as it highly prefers to exchange the alcohol moiety of the ester molecule with another free alcohol. This discovery exposes the limitations for using Eat1 as an AAT but also provides insights into the portfolio of ester compounds produced by many yeasts.An alternative approach for microbial ester production is described in Chapter 4. This approach uses the BMCs of C. beijerinckii NCIMB 8052 to encapsulate an AAT enzyme and produce propyl propionate as terminal product. As a proof of principle, the GFP protein is encapsulated in the BMCs by using N-terminal EPs. Unfortunately, due to time restrictions, the encapsulation of AATs and the successful utilization of C. beijerinckii BMCs to produce propyl propionate was not (yet) realized.Chapter 5 describes the development of a simple genome engineering tool for multiplex gene knock-out in C. beijerinckii NCIMB 8052. This tool is based on the CRISPR-FnCas12a system which is only expressed in the presence of the xylose inducer. 25-100% knock-out efficiency was observed when targeting single genes for deletion whereas, 18% knock-out efficiency was apparent when targeting two genes for deletion. The spacer sequence and spacer order in a CRISPR array is also assessed, showing that it highly influences the knock-out efficiency.Low HR efficiency along with strong CRISPR-Cas counterselection limit the application of CRISPR-Cas genome engineering tools in non-model prokaryotes. In Chapter 6, these limitations are mitigated through the development of SIBR. SIBR was developed and used as a widely applicable gene control system that can control the expression of virtually any gene of interest in any mesophilic prokaryote of interest. SIBR was used to control the expression of the FnCas12a protein (collectively termed SIBR-Cas) and allow more time for HR to occur before induction of counterselection. SIBR-Cas was applied in three different bacteria with high editing efficiencies demonstrating the wide applicability of SIBR.