Flux optimization using multiple promoters in Halomonas bluephagenesis as a model chassis of the next generation industrial biotechnology.

Predictability and robustness are challenges for bioproduction because of the unstable intracellular synthetic activities. With the deeper understanding of the gene expression process, fine-tuning has become a meaningful tool for biosynthesis optimization. This study characterized several gene expression elements and constructed a multiple inducible system that responds to ten different small chemical inducers in halophile bacterium Halomonas bluephagenesis. Genome insertion of regulators was conducted for the purpose of gene cluster stabilization and regulatory plasmid simplification. Additionally, dynamic ranges of the multiple inducible systems were tuned by promoter sequence mutations to achieve diverse scopes for high-resolution gene expression control. The multiple inducible system was successfully employed to precisely control chromoprotein expression, lycopene and poly-3-hydroxybutyrate (PHB) biosynthesis, resulting in colorful bacterial pictures, optimized cell growth, lycopene and PHB accumulation. This study demonstrates a desirable approach for fine-tuning of rational and efficient gene expressions, displaying the significance for metabolic pathway optimization.

Operational reliability of urban drainage systems under uncertainties.

Water quality risks from overflows have attracted significant research attention, and the reliability of urban drainage systems (UDS) is in urgent need of assessment and improvement. The overflow volume and concentration of critical pollutants are generally used as assessment indicators, which is quite time consuming and cumbersome especially under continuous rainfall. Simplifying the water quality risk assessment indicators for the UDS reliability is intractable. For this purpose, this study proposes the detention tank emptying time as a new reliability evaluation indicator, which greatly reduces the calculation burden by converting water quality risk into hydraulic risk. On this basis, the effects of rainfall, dry weather flow (DWF), actuators and their interactions on reliability are quantified by massive scenarios. It shows that the DWF affects the emptying process via weekly and daily seasonality and its interaction with rainfall is mainly responsible for unreliability. Further, the engineering facility linkage controlled by the actuator to cope with the interaction is the key. Particularly, the Prophet algorithm is innovatively applied to mine the patterns and generate the DWF series for the challenge of sparse DWF data. In conclusion, the indicator proposed expands the connotation of UDS reliability assessment, prompting a small investment in replacing actuators with better controllability and greatly improving reliability. It guides the engineering planning and enhancement from a new perspective of whole-chain optimization from the global to the detailed level.

Engineering an oleic acid-induced system for Halomonas, E. coli and Pseudomonas.

Ligand-induced system plays an important role for microbial engineering due to its tunable gene expression control over timings and levels. An oleic acid (OA)-induced system was recently constructed based on protein FadR, a transcriptional regulator involved in fatty acids metabolism, for metabolic control in Escherichia coli. In this study, we constructed a synthetic FadR-based OA-induced systems in Halomonas bluephagenesis by hybridizing the porin promoter core region and FadR-binding operator (fadO). The dynamic control range was optimized over 150-fold, and expression leakage was significantly reduced by tuning FadR expression and positioning fadO, forming a series of OA-induced systems with various expression strengths, respectively. Additionally, ligand orthogonality and cross-species portability were also studied and showed highly linear correlation among Halomonas spp., Escherichia coli and Pseudomonas spp. Finally, OA-induced systems with medium- and small-dynamic control ranges were employed to dynamically control the expression levels of morphology associated gene minCD, and monomer precursor 4-hydroxybutyrate-CoA (4HB-CoA) synthesis pathway for polyhydroxyalkanoates (PHA), respectively, in the presence of oleic acid as an inducer. As a result, over 10 g/L of poly-3-hydroxybutyrate (PHB) accumulated by elongated cell sizes, and 6 g/L of P(3HB-co-9.57 mol% 4HB) were obtained by controlling the dose and induction time of oleic acid only. This study provides a systematic approach for ligand-induced system engineering, and demonstrates an alternative genetic tool for dynamic control of industrial biotechnology.

Tailor-Made Polyhydroxyalkanoates by Reconstructing Pseudomonas Entomophila.

Microbial polyhydroxyalkanoates (PHA) containing short- and medium/long-chain-length monomers, abbreviated as SCL-co-MCL/LCL PHAs, generate suitable thermal and mechanical properties. However, SCL-co-MCL/LCL PHAs with carbon chain longer than nine are difficult to synthesize due to the low specificity of PHA synthase PhaC and the lack of either SCL- or MCL/LCL monomer precursor fluxes. This study succeeds in reprogramming a ß-oxidation weakened Pseudomonas entomophila containing synthesis pathways of SCL 3-hydroxybutyryl-CoA (3HB) from glucose and MCL/LCL 3-hydroxyalkanoyl-CoA from fatty acids with carbon chain lengths from 9 to 18, respectively, that are polymerized under a low specificity PhaC61-3 to form P(3HB-co-MCL/LCL 3HA) copolymers. Through rational flux-tuning approaches, the optimized recombinant P. entomophila accumulates 55 wt% poly-3-hydroxybutyrate in 8.4 g L-1 cell dry weight. Combined with weakened ß-oxidation, a series of novel P(3HB-co-MCL/LCL 3HA) copolymers with over 60 wt% PHA in 9 g L-1 cell dry weight have been synthesized for the first time. P. entomophila has become a high-performing platform to generate tailor-made new SCL-co-MCL/LCL PHAs.

Effects of Yttrium Addition on the Microstructure Evolution and Electrochemical Corrosion of SN-9Zn Lead-Free Solders Alloy.

Electrochemical corrosion behavior of ternary tin-zinc-yttrium (Sn-9Zn-xY) solder alloys were investigated in aerated 3.5 wt.% NaCl solution using potentiodynamic polarization techniques, and the microstructure evolution was obtained by scanning electron microscope (SEM). Eight different compositions of Sn-9Zn-xY (x = 0, 0.02, 0.04, 0.06, 0.08, 0.10, 0.20, and 0.30 wt.%) were compared by melting. The experimental results show that when the content of Y reached 0.06 wt.%, the grain size of Zn-rich phase became the smallest and the effect of grain refinement was the best, but there was no significant effect on the melting point. With the increases of Y content, the spreading ratio first increased and then decreased. When the content of Y was 0.06 wt.%, the Sn-9Zn-0.06Y solder alloy had the best wettability on the Cu substrate, which was increased by approximately 20% compared with Sn-9Zn. Besides, the electrochemical corrosion experimental shows that the Y can improve the corrosion resistance of Sn-9Zn system in 3.5 wt.% NaCl solution, and the corrosion resistance of the alloy is better when the amount of Y added is larger within 0.02-0.30 wt.%. Overall considering all performances, the optimal performance can be obtained when the addition amount of Y is 0.06.

Reversible thermal regulation for bifunctional dynamic control of gene expression in Escherichia coli.

Genetically programmed circuits allowing bifunctional dynamic regulation of enzyme expression have far-reaching significances for various bio-manufactural purposes. However, building a bio-switch with a post log-phase response and reversibility during scale-up bioprocesses is still a challenge in metabolic engineering due to the lack of robustness. Here, we report a robust thermosensitive bio-switch that enables stringent bidirectional control of gene expression over time and levels in living cells. Based on the bio-switch, we obtain tree ring-like colonies with spatially distributed patterns and transformer cells shifting among spherical-, rod- and fiber-shapes of the engineered Escherichia coli. Moreover, fed-batch fermentations of recombinant E. coli are conducted to obtain ordered assembly of tailor-made biopolymers polyhydroxyalkanoates including diblock- and random-copolymer, composed of 3-hydroxybutyrate and 4-hydroxybutyrate with controllable monomer molar fraction. This study demonstrates the possibility of well-organized, chemosynthesis-like block polymerization on a molecular scale by reprogrammed microbes, exemplifying the versatility of thermo-response control for various practical uses.

Engineering Halomonas bluephagenesis as a chassis for bioproduction from starch.

Halomonas bluephagenesis has been successfully engineered to produce multiple products under open unsterile conditions utilizing costly glucose as the carbon source. It would be highly interesting to investigate if H. bluephagenesis, a chassis for the Next Generation Industrial Biotechnology (NGIB), can be reconstructed to become an extracellular hydrolytic enzyme producer replacing traditional enzyme producer Bacillus spp. If successful, cost of bulk hydrolytic enzymes such as amylase and protease, can be significantly reduced due to the contamination resistant and robust growth of H. bluephagenesis. This also allows H. bluephagenesis to be able to grow on low cost substrates such as starch. The modularized secretion machinery was constructed and fine-tuned in H. bluephagenesis using codon-optimized gene encoding α-amylase from Bacillus lichenifomis. Screening of suitable signal peptides and linkers based on super-fold green fluorescence protein (sfGFP) for enhanced expression in H. bluephagenesis resulted in a 7-fold enhancement of sfGFP secretion in the recombinant H. bluephagenesis. When the gene encoding sfGFP was replaced by α-amylase encoding gene, recombinant H. bluephagenesis harboring this amylase secretory system was able to produce poly(3-hydroxybutyrate) (PHB), poly(3-hydroxybutyrate-co-4-hydroxybutyrate) (P34HB), poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV), ectoine and L-threonine utilizing starch as the growth substrate, respectively. Recombinant H. bluephagenesis TN04 expressing genes encoding α-amylase and glucosidase on chromosome and plasmid-based systems, respectively, was able to grow on corn starch to approximately 10 g/L cell dry weight containing 51% PHB when grown in shake flasks. H. bluephagenesis was demonstrated to be a chassis for productions of extracellular enzymes and multiple products from low cost corn starch.