Suggested reaction knockouts by cMCS.

The engineered pathway connecting MR degradation and ACT biosynthesis is highlighted in green. Knockouts in strain design 1: red cross; 2: blue cross; 4: orange cross; and 5: purple cross.</p

cMCS computation results.

Includes results of cMCS computations on GLC, MR, GLC + MR and six other aromatic substrates. (XLSX)</p

cMCS-suggested strain designs.

Fermentation-based biosynthesis in synthetic biology relies heavily on sugar-derived feedstocks, a limited and carbon-intensive commodity. Unconventional feedstocks from less-noble sources such as waste are being utilized to produce high-value chemical products. Azo dyes, a major pollutant commonly discharged by food, textile, and pharmaceutical industries, present significant health and environmental risks. We explore the potential of engineering Pseudomonas putida KT2440 to utilize azo dyes as a substrate to produce a polyketide, actinorhodin (ACT). Using the constrained minimal cut sets (cMCS) approach, we identified metabolic interventions that optimize ACT biosynthesis and compare the growth-coupling solutions attainable on an azo dye compared to glucose. Our results predicted that azo dyes could perform better as a feedstock for ACT biosynthesis than glucose as it allowed growth-coupling regimes that are unfeasible with glucose and generated an 18.28% higher maximum ACT flux. By examining the flux distributions enabled in different carbon sources, we observed that carbon fluxes from aromatic compounds like azo dyes have a unique capability to leverage gluconeogenesis to support both growth and production of secondary metabolites that produce excess NADH. Carbon sources are commonly chosen based on the host organism, availability, cost, and environmental implications. We demonstrated that careful selection of carbon sources is also crucial to ensure that the resulting flux distribution is suitable for further metabolic engineering of microbial cell factories.</div

Cofactor turnover rates in iJN1462c measured by their metabolite flux-sum.

Note that the difference in cell objective and carbon source affects cofactor flux-sum.</p

Production envelopes mapping feasible flux ranges calculated through FVA.

(A) MRt1 vs BIOMASS_KT2440_WT3; MR. (B) MRt1 vs BIOMASS_KT2440_WT3; GLC+MR. (C) MRt1 vs ACTt; MR. (D) MRt1 vs ACTt; GLC+MR. (E) ACTt vs BIOMASS_KT2440_WT3; MR. (F) ACTt vs BIOMASS_KT2440_WT3; GLC+MR. (G) ACTt vs BIOMASS_KT2440_WT3; GLC. (H) ACTt vs Biomass; iAA1259 S. coelicolor model with GLC.</p

Effect of Corn Steep Liquor on Bamboo Biochemical Pulping Using Phanerochaete chrysosporium

The effect of corn steep liquor (CSL) loading on white rot fungi inoculums in the biochemical pulping of betung bamboo was investigated. The best condition of the pretreatment was also determined. There were two conditions of CSL amount used i.e. 1% (v/w) and 10 % (v/w) of CSL used in 100 ml of inoculums. In short, fresh bamboo chips without bark was incubated with Phanaerochaete chrysosporium inoculum stocks for 30 and 45 days at room temperature and followed by Kraft and Soda pulpings. SEM images of pretreated chips were taken after incubation. The pulp yield, Kappa number, freeness, delignification selectivity, and brightness were analyzed. The more CSL amount added with Kraft process gave better results on pulp yield. Increasing incubation time increased pulp yields and decreased the Kappa numbers. On the other hand, freeness and brightness of pulp increased especially for Kraft process. The SEM images showed that there were cell walls degradation activities. The best properties of pulp were obtained by the Kraft process for the treatment of 10% CSL with 30 days of incubation time.Key words: betung bamboo, biochemical pulping, corn steep liquor, Phanaerochaete chrysosporiu

OptKnock, OptGene, and cMCS strain design suggestions.

Shows the production envelopes of strain designs generated by OptKnock, OptGene, and cMCS. (DOCX)</p

Simulation on other aromatic carbon sources.

Evaluation of the strain designs found by the cMCS computation on MR in other common aromatic carbon sources. (DOCX)</p

Flux distributions through key metabolites in the aromatic catabolism pathway (acetaldehyde), ED pathway, PP pathway, and the glyoxylate and glycerate shunt in iJN1462c and suggested growth-coupled strain designs.

Calculated in in silico M9 Minimal Medium with MR or GLC as the sole carbon source. iJN1462c was observed twice in different optimization objectives: biomass and ACT production; all growth-coupled designs were observed on MR only and were optimized for biomass/growth.</p

Simplified map of the catechol degradation pathways, ACT biosynthesis, and central carbon metabolism in iJN1462c.

The engineered pathway connecting MR degradation and ACT biosynthesis is highlighted in green.</p