Microbial production of hyaluronic acid (HA) is an attractive substitute for extraction of this biopolymer from animal tissues. Natural producers such as Streptococcus zooepidemicus are potential pathogens; therefore, production of HA by recombinant bacteria that are generally recognized as safe (GRAS) organisms is a viable alternative that is being extensively explored. However, plasmid-based expression systems for HA production by recombinant bacteria have the inherent disadvantage of reduced productivity because of plasmid instability. To overcome this problem, the HA synthesis genes (hasA-hasB and hasA-hasB-hasC) from has-operon of S. zooepidemicus were integrated into the chromosome of Lactococcus lactis by site-directed, double-homologous recombination developing strains VRJ2AB and VRJ3ABC. The chromosomal integration stabilized the genes and obviated the instability observed in plasmid-expressed recombinant strains. The genome-integrated strains produced higher molecular weight (3.5-4 million Dalton [MDa]) HA compared to the plasmid-expressed strains (2 MDa). High molecular weight HA was produced when the intracellular concentration of uridine diphosphate N-acetylglucosamine (UDP-GlcNAc) and uridine diphosphate-glucuronic acid (UDP-GlcUA) was almost equal and hasA to hasB ratio was low. This work suggests an optimal approach to obtain high molecular weight HA in recombinant strains.

1 5 1 6The E. coli metabolome is an interconnected set of enzymes that has measurable kinetic parameters ascribed for 1 7 the production of most of its metabolites. Flux Balance Analysis (FBA) or Ordinary Differential Equation 1 8 (ODE) models are used to increase product yield using defined media. However, they either give a range (FBA) 1 9 or exact amount (ODE) of metabolite yield which isn't true as the transcriptome diversity of individual cells 2 0 isn't considered. We formulate the metabolic-behaviour of individual cells by using a POpulation SYstems- 1Biology ALgorithm (POSYBAL) which predicts multiple-gene knockouts for increasing industrially relevant 2 2 metabolites. We validate this prediction for producing isobutanol (Heterogenous metabolite) and shikimate 2 3 (Homogenous metabolite) where, the product-yield was increased by 40 times (~2000 ppm) and 42 times 2 4 (~3000 ppm) respectively. Also, we introduce a nitrogen-swap in standard media to its low-nitrogen counterpart 2 5 during post-growth phase to redistribute flux towards non-nitrogenous pathways for increasing overall product-2 6 yield. Further, our model shows growth-phase diversity in bacterial population even under normal glucose-2 7 uptake, portraying a real-world scenario of diverse and robust environment thus, making it evolutionarily 2 8 favourable to threats such as anti-bacterial attack.2 9 3 0

Flux balance analysis (FBA) and ordinary differential equation models have been instrumental in depicting the metabolic functioning of a cell. Nevertheless, they demonstrate a population’s average behavior (summation of individuals), thereby portraying homogeneity. However, living organisms such as Escherichia coli contain more biochemical reactions than engaging metabolites, making them an underdetermined and degenerate system. This results in a heterogeneous population with varying metabolic patterns. We have formulated a population systems biology model that predicts this degeneracy by emulating a diverse metabolic makeup with unique biochemical signatures. The model mimics the universally accepted experimental view that a subpopulation of bacteria, even under normal growth conditions, renders a unique biochemical state, leading to the synthesis of metabolites and persister progenitors of antibiotic resistance and biofilms. We validate the platform’s predictions by producing commercially important heterologous (isobutanol) and homologous (shikimate) metabolites. The predicted fluxes are tested in vitro resulting in 32- and 42-fold increased product of isobutanol and shikimate, respectively. Moreover, we authenticate the platform by mimicking a bacterial population in the presence of glyphosate, a metabolic pathway inhibitor. Here, we observe a fraction of subsisting persisters despite inhibition, thus affirming the signature of a heterogeneous populace. The platform has multiple uses based on the disposition of the user.