Technical-Economical Approach for PHB Production by Ralstonia Eutropha Strain Using Concentrated Vinasse as Carbon Source and Other Biotechnological Applications

The Brazilian ethanol industry is one of the most important in the global market, however these important industrial activities have been generating significant amounts of vinasse and its management has become costly for distilleries. In this study, the aim was to evaluate concentrated and in natura vinasse as basal culture media for biotechnological processes. Different bacteria and processes were assessed: L-threonine production by E. coli THR14, with glucose as carbon source; PHB production by halophilic strain Halomonas sp. HG03, with sucrose as carbon source; and PHB biosynthesis by R. eutropha L359PCJ, which used glycerol from vinasse as carbon source. Strains were evaluated firstly in shake flasks cultivations using vinasse-based media. E. coli THR14 had no statistical difference for biomass and L-threonine concentrations among control and vinasse-based treatments (up to 50% v v-1 of in natura vinasse). Halomonas sp. HG03 and R. eutropha L359PCJ were cultivated in mineral media diluted by in natura (50% and 75% v v-1) and concentrated (50% and 75% v v-1) vinasses. Higher vinasse concentrations resulted in higher cellular growth rather than PHB accumulation for both bacteria. In vinasse-based treatments, Halomonas sp. HG03 had PHB content between 19.6 – 75.2% and R. eutropha L359PCJ, 48.4 – 68.5%. 50% (v v-1) of concentrated vinasse was the most attractive condition for PHB production by both bacteria. Further experiments in CSTR bioreactors used this nutritional condition and R. eutropha L359PCJ had PHB content of 66.3%, concentrations of residual cell dry weight (rCDW) = 9.4 g L-1 and PHB = 18.6 g L-1, with YX/S = 0.16 g gGLYCEROL-1, YP/S = 0.32 g gGLYCEROL-1 and 0.25 gPHB Lh-1. Halomonas sp. HG03 had PHB content of 45.7%, rCDW = 9.8 g L-1, PHB = 8.3 g L-1 and YX/S = 0.18 g gSUCROSE-1, YP/S = 0.16 g gSUCROSE-1 and 0.12 gPHB Lh-1. Finally, cost reductions of PHB production by R. eutropha L359PCJ with concentrated vinasse-based medium were evaluated in silico by using SuperPro Designer. As a partial source of glycerol and other nutrients for PHB production by R. eutropha L359PCJ, vinasse reduced overall production costs by 13%. Simulated processes that used concentrated vinasse-based media combined with improvements of PHB productivity and higher cellular densities had production costs between US$ 3.9 – 7.5/kgPHB and 2.6 – 7.3 years of payback time.

Improving l-threonine production in Escherichia coli by elimination of transporters ProP and ProVWX

Background Betaine, an osmoprotective compatible solute, has been used to improve l-threonine production in engineered Escherichia colil-threonine producer. Betaine supplementation upregulates the expression of zwf encoding glucose-6-phosphate dehydrogenase, leading to the increase of NADPH, which is beneficial for l-threonine production. In E. coli, betaine can be taken through ProP encoded by proP or ProVWX encoded by proVWX. ProP is a H+-osmolyte symporter, whereas ProVWX is an ABC transporter. ProP and ProVWX mediate osmotic stress protection by transporting zwitterionic osmolytes, including glycine betaine. Betaine can also be synthesized in E. coli by enzymes encoded by betABIT. However, the influence of ProP, ProVWX and betABIT on l-threonine production in E. coli has not been investigated. Results In this study, the influence of ProP, ProVWX and betABIT on l-threonine production in E. coli has been investigated. Addition of betaine slightly improved the growth of the l-threonine producing E. coli strain TWF001 as well as the l-threonine production. Deletion of betABIT retarded the growth of TWF001 and slightly decreased the l-threonine production. However, deletion of proP or/and proVWX significantly increased the l-threonine production. When proP was deleted, the l-threonine production increased 33.3%; when proVWX was deleted, the l-threonine production increased 40.0%. When both proP and proVWX were deleted, the resulting strain TSW003 produced 23.5 g/l l-threonine after 36 h flask cultivation. The genes betABIT, proC, fadR, crr and ptsG were individually deleted from TSW003, and it was found that further absence of either crr (TWS008) or ptsG (TWS009) improved l-threonine production. TSW008 produced 24.9 g/l l-threonine after 36 h flask cultivation with a yield of 0.62 g/g glucose and a productivity of 0.69 g/l/h. TSW009 produced 26 g/l l-threonine after 48 h flask cultivation with a yield of 0.65 g/g glucose and a productivity of 0.54 g/l/h, which is 116% increase compared to the control TWF001. Conclusions In this study, l-threonine-producing E. coli strains TSW008 and TSW009 with high l-threonine productivity were developed by regulating the intracellular osmotic pressure. This strategy could be used to improve the production of other products in microorganisms.

Use of an Alternative Pathway for the Synthesis of Isoleucine in Escherichia coli Threonine-Producing Strains

An E. coli strain in which all known pathways of threonine catabolism were inactivated (Δtdh, ΔltaE, ΔAilvA, ΔtdcB, AyiaY) has been constructed. The possibility of an alternative pathway for the isoleucine synthesis by expressing heterologous citramalate synthase from Leptospira interrogans in an E. coli strain carrying the ΔilvA deletion was demonstrated. It was observed that the cimA overexpression has a negative effect on the threonine production. We developed a system for regulated gene expression based on the inducible promoter PLtetO and TetR repressor of the tetracycline operon. A threonine producing strain B-1201 in which the cimA gene is expressed under the control of the regulated promoter was constructed. A correlation of the threonine productivity and the expression level of the cimA gene was shown by culturing the B-1201 strain in fermenter. The optimal inductor content for the maximum threonine accumulation was also determined. Escherichia coli, strain, threonine, citramalate synthase This work was supported by the Ministry of Education and Science of the Russian Federation (project code RFMEFI61017X0011), and it was carried out using the equipment of the National Bio-Resource Center «All-Russian Collection of Industrial Microorganisms», NRC «Kurchatov Institute» - GOSNIIGENETIKA. The authors are grateful to Dr. I. V. Manukhov for providing the MG1655Z1strain and pZE21-lux plasmid, and for teaching techniques of bioluminescence measuring.