Rationally Engineered Synthetic Coculture for Improved Biomass and Product Formation

<div><p>In microbial ecosystems, bacteria are dependent on dynamic interspecific interactions related to carbon and energy flow. Substrates and end-metabolites are rapidly converted to other compounds, which protects the community from high concentrations of inhibitory molecules. In biotechnological applications, pure cultures are preferred because of the more straight-forward metabolic engineering and bioprocess control. However, the accumulation of unwanted side products can limit the cell growth and process efficiency. In this study, a rationally engineered coculture with a carbon channeling system was constructed using two well-characterized model strains <i>Escherichia coli</i> K12 and <i>Acinetobacter baylyi</i> ADP1. The directed carbon flow resulted in efficient acetate removal, and the coculture showed symbiotic nature in terms of substrate utilization and growth. Recombinant protein production was used as a proof-of-principle example to demonstrate the coculture utility and the effects on product formation. As a result, the biomass and recombinant protein titers of <i>E. coli</i> were enhanced in both minimal and rich medium simple batch cocultures. Finally, harnessing both the strains to the production resulted in enhanced recombinant protein titers. The study demonstrates the potential of rationally engineered cocultures for synthetic biology applications.</p></div

Biomasses and fluorescence of <i>E. coli</i> expressing sfGFP/pAK400c (ECsf) and <i>A. baylyi</i> ADP1Δ<i>gntT::Kan^r/tdk</i> expressing empty plasmid pBAV1C-<i>ara</i> (ABc) in mono and cocultures.

<p>Fluorescence is produced solely by <i>E. coli</i> expressing GFP. The strains were cultivated in a minimal salts medium supplemented with 50; 100; or 250; mM glucose for 24 h. A) Biomasses (optical density, OD₆₀₀) of ECsf monocultures and cocultures (ECsfABc). B) Fluorescence signals measured for monocultures and cocultures demonstrating the production of recombinant protein in cultures. The mean and standard deviation of two independent cultures are shown.</p

The proposed carbon flow in the wild type <i>Escherichia coli</i> culture and in the coculture of engineered <i>Acinetobacter baylyi</i> ADP1 and <i>E. coli</i>.

<p>A) <i>E. coli</i> culture supplied with excess glucose readily shifts to an overflow metabolism (*), producing large amounts of acetate into the culture medium. Acetate inhibits growth and reduces product formation, and carbon flow is directed off the product route. B) In a coculture involving directed carbon flow, the strain <i>A. baylyi</i> ADP1 is made deficient in glucose utilization by a knock-out of gluconate permease <i>gntT</i> and is solely dependent on the end-metabolites (acetate) of <i>E. coli</i>. Carbon is further metabolized and can be directed to biomass and the product of interest. Metabolic pathways in the figure are simplified and only the main products are shown.</p

Monitored bioreactor cultivations of the monoculture ECg and the coculture ECgABg.

<p>Both strains produce GFP by expressing the same genetic construct pBAV1C-T5-GFP. The cultivations were performed in a bioreactor in a rich medium supplied with 100 mM glucose and aeration. A) Culture parameters; pH (large circles) and oxygen partial pressure (pO2 %, small circles) for ECg monoculture (empty circles) and ECgABg coculture (filled circles) B) Total biomasses (OD₆₀₀) of the cultures and proportion of ABg cells (%, CFU) in the coculture. C) Fluorescence signals (cts) for the monoculture ECg and coculture ECgABg demonstrating the production of recombinant protein in the cultures. The mean and standard deviation of 2–4 independent culture samples are shown. ECg - <i>E. coli</i> expressing pBAV1C-T5-GFP, ABg - <i>A. baylyi ADP1ΔgntT::Kan^r/tdk</i> expressing pBAV1C-T5-GFP.</p

The total biomass of the coculture of <i>E. coli</i> and ADP1Δ<i>gntT</i>::<i>Kan^r/tdk</i>[<i>lux_tet^r</i>] (ABlux), and the proportion of ABlux in the total biomass.

<p>The strain ABlux is deficient in utilizing glucose. During the cultivation, the growth of ABlux was monitored in real-time via a luminescence reporter <i>luxCDABE</i> (the inlet). The cells were cultivated in a minimal salts medium supplied with glucose for 12 h. The mean and standard deviation of the two independent cultures are shown. Note the logarithmic scale in biomass and luminescence y-axes. Total biomass – line with squares; proportion of ABlux cells in total biomass – columns.</p

Fluorescent Protein Based FRET Pairs with Improved Dynamic Range for Fluorescence Lifetime Measurements

<div><p>Fluorescence Resonance Energy Transfer (FRET) using fluorescent protein variants is widely used to study biochemical processes in living cells. FRET detection by fluorescence lifetime measurements is the most direct and robust method to measure FRET. The traditional cyan-yellow fluorescent protein based FRET pairs are getting replaced by green-red fluorescent protein variants. The green-red pair enables excitation at a longer wavelength which reduces cellular autofluorescence and phototoxicity while monitoring FRET. Despite the advances in FRET based sensors, the low FRET efficiency and dynamic range still complicates their use in cell biology and high throughput screening. In this paper, we utilized the higher lifetime of NowGFP and screened red fluorescent protein variants to develop FRET pairs with high dynamic range and FRET efficiency. The FRET variations were analyzed by proteolytic activity and detected by steady-state and time-resolved measurements. Based on the results, NowGFP-tdTomato and NowGFP-mRuby2 have shown high potentials as FRET pairs with large fluorescence lifetime dynamic range. The <i>in vitro</i> measurements revealed that the NowGFP-tdTomato has the highest Förster radius for any fluorescent protein based FRET pairs yet used in biological studies. The developed FRET pairs will be useful for designing FRET based sensors and studies employing Fluorescence Lifetime Imaging Microscopy (FLIM).</p></div

Variations in the FRET response of FRET pairs on proteolytic cleavage over time.

<p>The control is NowGFP-mRuby2 FRET pair without thrombin cleavage site (LVPS instead of LVPR). ΔR/R was computed as (R₀-R_F)/R₀ where R is donor:acceptor ratio and R₀ is the donor:acceptor ratio when there is no FRET and R_F is the FRET ratio</p

Emission spectra of the FRET constructs.

<p>Fluorescence emission spectrum (at 480 nm excitation) of the FRET constructs treated with thrombin. The schematics of the FRET constructs are displayed above the spectrum. "LVPR" represents the sequence GGGSLVPRGS. The decrease in the FRET in time as a result of proteolytic cleavage can be observed from the spectrum. </p

SDS PAGE displaying the proteolytic activity.

<p>The absence of fusion protein band and the presence of the cleaved protein band is visible from Lane 2 and 4 confirming proteolytic cleavage. The dotted arrows indicate the cleaved product and the straight arrow indicate the fusion protein band. </p

Intracellular FLIM of <i>E</i>. <i>coli</i> cells.

<p><i>(A)</i> Fluorescence lifetime image of the cells displaying FRET. The cells are excited at 483 nm and the selective emission from the donor was monitored through band pass filter (510/20 nm). NowGFP is the cells expressing donor alone and the variation in lifetime as a result of FRET can be observed from the cells expressing the FRET pairs. The average lifetime is calculated from approximately 30 cells. Image size is 10 μm × 10 μm. <i>(B)</i> Fluorescence decay curve from the cells showing FRET. The decrease in the fluorescence lifetime due to FRET can be observed from the decay curve.</p