Origins of Cell-to-Cell Bioprocessing Diversity and Implications of the Extracellular Environment Revealed at the Single-Cell Level

Abstract

Bioprocess limitations imposed by microbial cell-to-cell phenotypic diversity remain poorly understood. To address this, we investigated the origins of such culture diversity during lipid production and assessed the impact of the fermentation microenvironment. We measured the single-cell lipid production dynamics in a time-invariant microfluidic environment and discovered that production is not monotonic, but rather sporadic with time. To characterize this, we introduce bioprocessing noise and identify its epigenetic origins. We linked such intracellular production fluctuations with cell-to-cell productivity diversity in culture. This unmasked the phenotypic diversity amplification by the culture microenvironment, a critical parameter in strain engineering as well as metabolic disease treatment.

Exploiting Bioprocessing Fluctuations to Elicit the Mechanistics of De Novo Lipogenesis in Yarrowia lipolytica

“…Alternatively, the rate limiting step in expressing or degrading lipids rests with the enzyme reaction rates, rather than the cytosolic substrate or product concentrations. This conclusion is in agreement with our previous finding that bioprocessing noise depends inversely on the intracellular lipid content: in essence, the bounded lipid flux (dS i /dt) enforces stronger (weaker) lipid content (S i ) fluctuations at low (high) cytosolic product concentrations [17]. …”

“…A recent report identified this form of heterogeneity as a non-heritable trait, as well as its protection role against lipotoxicity [11]. To a similar end, we observed at the single-cell level that cytosolic lipid accumulation is far from monotonic with time [17]. We identified this form of bioprocessing noise as the origin of the cell-to-cell heterogeneity, confirmed its epigenetic origins and dependence on the extracellular environment [17].…”

“…Following our previous analysis, the intracellular neutral lipid content ( S i , namely total LD area normalized over the cell area) was observed to fluctuate strongly over time [17]. The S i fluctuations were manifested both through fluctuations of the number of LDs ( N i ), as well as their average sizes ( α i − normalized over the cell size)–illustrated the longitudinal traces for 10 Y .…”

“…To a similar end, we observed at the single-cell level that cytosolic lipid accumulation is far from monotonic with time [17]. We identified this form of bioprocessing noise as the origin of the cell-to-cell heterogeneity, confirmed its epigenetic origins and dependence on the extracellular environment [17]. In addition to the cell-to-cell lipid content heterogeneity, another form of phenotypic heterogeneity persists in clonal populations, whereas some cells contain large-but-fewer LDs, and others contain small-but-more LDs.…”

“…Specifically, we selected the Po1g strain, which is transformed to overexpress the acetyl-CoA carboxylase (ACC) and diacylglycerol transferase (DGAT) genes, thereby allowing it to accumulate lipids [8]. By placing this strain within a microfluidic quasi-time invariant microenvironment–a crucial parameter in longitudinal analyses given the highly dynamic nature of the metabolome [26]–we previously reported the sporadic nature of lipid accumulation at early stationary phase [17]. Here, we employ these incessant fluctuations to statistically correlate the two phenotypes pertaining to large-but-few versus small-but-many LDs, and investigate the mechanistics of lipid accumulation and degradation under rich medium and protein translation inhibition steady-state conditions.…”