Flux-Balance Analysis and Mobile CRISPRi-Guided Deletion of a Conditionally Essential Gene in Shewanella oneidensis MR-1

Abstract

Carbon-neutral production of valuable bioproducts is critical to sustainable development but remains limited by the slow engineering of photosynthetic organisms. Improving existing synthetic biology tools to engineer model organisms to fix carbon dioxide is one route to overcoming the limitations of photosynthetic organisms. In this work, we describe a pipeline that enabled the deletion of a conditionally essential gene from the Shewanella oneidensis MR-1 genome. S. oneidensis is a simple bacterial host that could be used for electricity-driven conversion of carbon dioxide in the future with further genetic engineering. We used Flux Balance Analysis (FBA) to model carbon and energy flows in central metabolism and assess the effects of single and double gene deletions. We modeled the growth of deletion strains under several alternative conditions to identify substrates that restore viability to an otherwise lethal gene knockout. These predictions were tested in vivo using a Mobile-CRISPRi gene knockdown system. The information learned from FBA and knockdown experiments informed our strategy for gene deletion, allowing us to successfully delete an "expected essential" gene, gpmA. FBA predicted, knockdown experiments supported, and deletion confirmed that the "essential" gene gpmA is not needed for survival, dependent on the medium used. Removal of gpmA is a first step toward driving electrode-powered CO₂ fixation via RuBisCO. This work demonstrates the potential for broadening the scope of genetic engineering in S. oneidensis as a synthetic biology chassis. By combining computational analysis with a CRISPRi knockdown system in this way, one can systematically assess the impact of conditionally essential genes and use this knowledge to generate mutations previously thought unachievable.

Keywords: CRISPRi; Shewanella oneidensis; carbon metabolism; flux-balance analysis; genetic engineering; synthetic biology.

Figure 1

Central carbon metabolism of S. oneidensis MR-1. Metabolites are shown in green boxes, and the genes encoding the proteins which catalyze enzymatic reactions (black arrows) are shown in red. This schematic comprises components of glycolysis, the pentose phosphate pathway, and the TCA cycle. The targeted reaction catalyzed by the product of gpmA is in the yellow box. Gene names listed in red are as annotated in NCBI for S. oneidensis genome (NCBI:txid211586).

Figure 2

CRISPRi gene knockdown in S. oneidensis MR-1. Strains of S. oneidensis MR-1 were constructed to chromosomally express dCas9 and sgRNA targeting (A) nothing (nonessential gene control), (B) rpoC (essential gene control), or (C) gpmA. Both the dCas9 and sgRNA are under IPTG induction. Strains were pregrown in LB + selection at 30 °C. Three microliters of serial 10-fold dilutions of a 1.0 OD₆₀₀ cell suspension was plated on an LB +/– inducer (10 mM IPTG) as described in the Materials and Methods section. Each panel shows replicate (n = 3) plating of each strain.

Figure 3

CRISPRi knockdown of genes in S. oneidensis MR-1 with supplemented media. Previously constructed strains of MR-1, with sgRNA targeting either nothing (A) or gpmA (B), were pregrown as before. This time, strains were plated on LB + 10 mM IPTG supplemented with 10 mM uridine and 20 mM lactate. The growth rate of the gpmA knockdown was restored to WT-levels under these conditions. Each panel shows replicate (n = 3) plating of each strain.

Figure 4

Identification and aerobic growth of S. oneidensis WT vs ΔgpmA cells in LB. (A) PCR amplification of the gpmA locus from WT S. oneidensis MR-1 and a gpmA deletion strain obtained by homologous recombination under modified conditions. Primers were located approximately 500 bp upstream and downstream of the coding region of the 1.7 kb gene. (B) Overnight cultures were used to inoculate 500 μL of LB in a 48-well plate for a starting OD₆₀₀ of 0.1 in the wells. The plate was incubated with constant shaking at 30 °C in an aerobic plate reader, reading the OD₆₀₀ every 15 min. Lines represent the simple moving average of three biological replicates for WT cells (green, solid) and ΔgpmA cells (black, dashed), and gray ribbons represent standard deviation.

Figure 5

Experimental confirmation of growth capabilities of ΔgpmA cells grown in the minimal medium with various substrates. M5 medium was prepared as described in the Materials and Methods section. Growth of (A) ΔgpmA, and (B) WT with lactate (blue), the designated nucleoside (yellow), or a combination of the two (red) (lactate results are repeated in each graph). The ΔgpmA cultures were grown overnight aerobically in LB at 30 °C. Cells were normalized to an OD₆₀₀ of 1.0, and 500 μL of each was inoculated into M5 medium in a 48-well plate. The plate was incubated with constant shaking at 30 °C in a plate reader, measuring the OD₆₀₀ every 15 min. Lines represent the simple moving average of three biological replicates, and gray ribbons represent the standard deviation.

Figure 6

Metabolic strategy of MR-1 ΔgpmA cells. With the deletion of gpmA, metabolites entering the cell as a TCA cycle intermediate are cutoff from entering glycolysis to build biomass, and vice versa. To satisfy the cell requirements for growth, MR-1 ΔgpmA requires at least two carbon sources to grow; one entering “above” gpmA to build biomass (nucleoside) and one entering “below” gpmA to acquire energy and reducing equivalents (lactate).