(438g) Towards Engineering Robust Strains and Genetic Tools to Facilitate Bioproduction of Building-Block Chemicals from Diverse Feedstocks

Authors: 
Nielsen, D., Arizona State University
Machas, M., Arizona State University
Jones, C., Arizona State University
Microorganisms represent promising platforms for producing fuels, chemicals, and plastics, either directly or via hybrid processes. In many cases, however, their true potential remains limited by a number of factors, including end-product toxicity, limited substrate range, and a general lack of useful genetic tools. For example, aromatic chemicals represent an important class of petrochemicals which predominantly serve as precursors for a diverse range of synthetic applications, notably including plastic production. While the biosynthesis of diverse aromatic chemicals has been demonstrated, product toxicity remains a common challenge. To better understand the effects of aromatics exposure, the transcriptional response of Escherichia coli was determined by performing RNA sequencing (RNAseq) analysis using styrene as a severely inhibitory model compound. Moreover, the potential influence of the source of exposure was comparatively evaluated by applying RNAseq analysis to both styrene-producing and styrene-exposed cells. Overall, up-regulation of general stress regulators and several membrane-altering genes was observed, as well as response patterns suggesting that, in addition to the known effects of compromised membrane integrity, styrene may also inhibit cells via DNA damage. Next, to improve the aromatic production from biomass sugars, key mutations have been introduced into XylR (key regulator of the xylose-utilization system) to relieve the effects of carbon catabolite repression (CCR). In addition to enabling efficient co-utilization of both glucose and xylose in sugar mixtures, this approach promotes increased availability of key shikimic acid pathway precursors, leading to enhanced production. By introducing the XylR mutant into a phenylalanine over-producing strain, 3.2-fold higher phenylalanine titers and complete xylose consumption were possible when culturing using a 66% glucose/33% xylose mixture. Finally, metabolic engineering efforts typically require the ability to make multiple and successive genomic modifications in a single host. While this might be almost a trivial task for microbes such as E. coli, such is still not the case for cyanobacteria. Thus, as cyanobacteria continue to emerge as a popular chassis for sustainable chemical production, it is imperative that more efficient methods for their genome engineering be developed. We have developed a simple strategy for markerless strain construction in Synechococcus sp. PCC 7002 that utilizes a novel counterselection method. This method uniquely makes use of a growth essential gene as an integration site to transiently express those elements required for gene editing. The method is initially demonstrated using the CRE-loxP site-specific recombinase system and is now being extended for use with other gene editing methods and cyanobacterial strains.